JP3364972B2 - Transglutaminase gene from fish - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a DNA fragment having a gene encoding a polypeptide having a transglutaminase activity derived from fish, a recombinant plasmid incorporating the DNA, a transformant into which the plasmid is introduced, And a method for producing a polypeptide having a transglutaminase activity derived from fish, which comprises culturing the transformant.

[0002]

BACKGROUND OF THE INVENTION Transglutaminase is an enzyme that catalyzes the acyl transfer reaction of the γ-carboxamide group of the glutamine residue in the peptide chain. This transglutaminase acts intramolecularly and intermolecularly when the ε-amino group of the lysine residue in the protein acts as an acyl receptor.
A-(γ-Gln) -Lys cross-link is formed, and when a primary amine such as an amino acid or an amino acid derivative is present as an acyl acceptor, it is introduced into the protein. When water functions as an acyl acceptor, it is an enzyme that promotes a reaction in which glutamine residues are deamidated to become glutamic acid residues.

Transglutaminase is used for producing gel foods, gel cosmetics, yogurt, jelly, cheese and the like (Japanese Patent Publication No. 1-5038).
2). Furthermore, it is also used for producing heat-stable microcapsule materials, carriers for immobilized enzymes and the like.

As a transglutaminase derived from a microorganism, a calcium (Ca 2 +)-independent transglutaminase has been discovered from a bacterium belonging to the genus Streptoverticillium. As a specific example of the genus bacteria, Streptoverticillium griseocarneum (Streptoverticill
ium griseocarneum) IFO 12776, Streptoverticillium cinnamoneum sub sp. cinnamone
oneum) IFO 12852, Streptoverticillium mobaraense IFO1381
9 and the like (see JP-A-64-27471).

Further, transglutaminase derived from mammals is also known. For example, from guinea pig liver [Connellan, et al., Journal of Biolog
icalChemistry Vol. 246, No. 4, 1093-1098 (1971)], derived from human and bovine vascular endothelial cells [Gentile, et al.,
Journal of Biological Chemistry Volume 266, 478-483
(1991), and Nakanishi, et al., European Journal
of Biochemistry 202, 15-21 (1991)], human blood coagulation factor XIII [Takahashi, et al., Proc. Natl. A].
cad. Sci. USA 83, 8019-8023 (1986)].

[0006]

Conventional enzyme sources for industrial use of transglutaminase have been mammals and bacteria. However, the products of transglutaminase that we are familiar with on a daily basis are processed fish products such as kamaboko [Seki et al., NipponSuisan].
Gakkaishi 56, 125-132 (1990)], of course,
The transglutaminase acting on it is a fish-derived enzyme.

Examples of genetically modified transglutaminase for modifying food proteins include those derived from guinea pigs. The following items are conceivable as points that the transglutaminase derived from fish is superior in industrial use to the ones that are currently available and can be compared and examined.

[0008] A) Fish is an organism that humans have been eating for many years, even if they are raw. Therefore, it means that the transglutaminase present in this organism is also eating for many years, and it is of other biological origin. It can be said that there are no safety concerns compared with transglutaminase.

B) Regarding the difference in enzyme characteristics between fish and transglutaminase derived from other mammals, especially when considering application to fish paste products, first of all, the reactivity of the enzyme and the inactivation condition of the enzyme to act are considered. The influence on the production cost due to the difference (this will be described in the following example),
It is fully conceivable that there are differences in qualitative effects such as the natural texture of the reaction product and the texture (expression of elasticity). actually,
Fish transglutaminase was found to be highly reactive to fish actomyosin (see Examples).

[0010] C) The difference in the productivity of the gene recombinant transglutaminase when using a microorganism is also an important problem when considering the practical use of transglutaminase. Ikura et al. Have already reported on the production of guinea pig-derived transglutaminase using Escherichia coli (Reference, Eur. J. Biochem., Vol.187, 705-711, 1990).
However, the production amount was extremely low, that is, transglutaminase in the extract of the transglutaminase-producing bacterium was detectable using an antibody against the transglutaminase (about 2.6 mg per liter of medium). Considerable transglutaminase production). On the contrary,
The transglutaminase derived from this fish, for example, when produced in E. coli as a host, the enzyme
The productivity of S-soluble protein is equivalent to 10% to 15%, and the expression level is about 100 times or more that of guinea pig transglutaminase. The transglutaminase derived from this fish has a gene structure suitable for recombinant production. Is more likely to have. (Explained in the following examples)

The various characteristics of the above-mentioned fish-derived transglutaminase are obtained by applying the transglutaminase to industry,
Especially when considering modification of food proteins, it will be a great advantage. In addition, the shortage of marine resources due to the recent regulation of 200 nautical miles in the fishing area and the total catch limit has become a major problem due to the high cost of raw materials for processed marine products. When used in combination with protein, it is possible to reduce the concentration of surimi raw materials in processed products and increase the effective use of low-utilization fish resources.

On the other hand, in comparison with microbial-derived transglutaminase (hereinafter abbreviated as BTG), fish-derived transglutaminase is completely different in terms of structure and reaction mechanism. For example, the fact that BTG does not require calcium ions for the expression of the enzyme function may be different from the fish-derived transglutaminase obtained in the present invention, depending on the desired protein to be used. .

The following can be said in terms of differentiation from BTG.
It is possible that fish-derived transglutaminase can control the reaction time and reactivity by utilizing this calcium ion dependency when switching between when the enzyme is to react with a substrate and when the reaction is to be stopped. Also, BT
Compared with G, fish-derived transglutaminase has lower thermostability, so it can be heat-inactivated at a relatively low temperature.
This can be said to be advantageous for food processing that does not like high temperature heating.

The transglutaminase gene of fish has never been known. If it becomes possible to produce fish transglutaminase at low cost,
Not only will the industrial application range of transglutaminase be increased, but it will also be possible to provide more natural processed fish products using enzymes, and the production cost of these products can be reduced by reducing the concentration of raw material surimi used. . As described above, in order to establish an inexpensive supply system of fish-derived transglutaminase, there are problems such as gene acquisition, analysis, and expression of fish transglutaminase.

[0015]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors decided to obtain a DNA fragment having a gene encoding a fish transglutaminase and determine its nucleotide sequence. Successful. Based on this result, E. coli, Bacillus subtilis,
It became possible to express the gene using microorganisms such as yeast and mold, and it was possible to open the way to efficient mass production of fish-derived transglutaminase.

That is, the present invention is a DNA fragment encoding a polypeptide having transglutaminase activity derived from fish, a recombinant plasmid incorporating the DNA,
A transformant having the plasmid introduced therein, a method for producing the polypeptide, which comprises culturing the transformant,
And the polypeptide obtained by the production method.

As a method for obtaining a DNA fragment having a gene encoding a polypeptide having a fish-derived transglutaminase activity according to the present invention, the inventors of the present invention have synthesized a DNA fragment near the active center of guinea pig transglutaminase (control A method for isolating a target DNA fragment from cDNA by hybridization using the sequence table SEQ ID NO: 10) as a probe was adopted. Until now, with guinea pig transglutaminase itself or its gene,
There is no knowledge that the fish transglutaminase itself or its gene has high homology, and it is surprising that the target DNA fragment could be obtained by this method.

In addition to the above, the method of obtaining a DNA fragment having a gene encoding a polypeptide having a transglutaminase activity derived from fish according to the present invention includes the following. A) A polypeptide having a transglutaminase activity derived from fish is isolated and purified, and the entire base sequence is chemically synthesized based on the determined amino acid sequence.
B) A part of the nucleotide sequence is synthesized based on the determined amino acid sequence, and this is used as a probe for cloning from a cDNA bank or genomic bank. The cloning method may be a hybridization method, but PCR
The method may be used. C) or mRNA is subjected to an in vitro translation system of white germ or rabbit reticulocyte to determine a fraction in which mRNA encoding a polypeptide having transglutaminase activity is present,
A desired cDNA fragment can be prepared and obtained from there.

The DNA fragment having a gene encoding a polypeptide having a transglutaminase activity derived from fish according to the present invention includes SEQ ID NO: 2 in Sequence Listing,
Examples include those encoding the polypeptides described in 3, 6, 7, 43, and 44.

Such a DNA fragment can include various base sequences in consideration of the degeneracy of gene codons. Those base sequences can be easily selected by those skilled in the art by various elements of the gene expression system, for example, the use of preferential codons according to the type of host cell, avoidance of higher-order structure formed in transcribed RNA, etc. I will get it. As a specific example, an example of a natural base sequence is shown in SEQ ID NOs: 4, 8, and 45 in the sequence listing, and an example in which a part of the natural base sequence is artificially converted is shown in SEQ ID NOS: 12, 13, 14, 15, 16,
Shown at 17, 18, and 19. Further, such a DNA base sequence may be one cloned from nature or artificially chemically synthesized DNA.

Further, such a DNA fragment has mutations due to individual differences in fish and multiple copies of genes, and mutations due to substitution, deletion, insertion, etc. of nucleotide sequences due to differences in organs and tissues. , Which may include essentially equivalent DNA fragments that are still capable of expressing transglutaminase activity. Their presence is described in the examples below.

The present invention also makes it possible to supply fish-derived transglutaminase by expressing it by a microorganism modified by a gene recombination technique. The recombinant plasmid used at this time is a known expression vector corresponding to the desired expression system, and a DNA fragment having a gene encoding fish-derived transglutaminase is added by a conventionally known method.
It can be prepared by inserting. As an expression vector for E. coli, an expression vector plasmid (Invitrogen, trade name: Xpress SystemT) that highly expresses a fusion protein of T7gene10 and a linker peptide is used.
M) and expression vector plasmids (Pharmacia LKB: trade name pGEK-based plasmids) that highly express fusion proteins with glutathione-S-transferase. A more preferred expression vector is pBSF2-SD
7, pT13sNco. On the other hand, as an expression vector in baker's yeast, there is pYES2 (INVITROGEN) in which the promoter of GAL1 which is a gene encoding galactokinase can be used for expressing a foreign gene.

The present invention also relates to various transformed transformants obtained by introducing an expression vector carrying a transglutaminase gene. Organisms that can be transformants include prokaryotic cells such as Escherichia coli and Bacillus subtilis, and yeast, eukaryotic cells such as mold, but more preferable organisms are Escherichia coli and yeast, more preferably E. coli HB101 strain and Saccharomyces. cerevisiae
It is INVSC2 strain. These transformants allow the introduced transglutaminase gene to produce and accumulate the enzyme intracellularly or in the medium.

Finally, the present invention relates to a method for producing a polypeptide having transglutaminase activity by culturing the above transformant. The culture conditions are
It can be appropriately determined by those skilled in the art according to the type of transformant. In addition, the enzyme expressed and accumulated in cells or secreted into the medium can be isolated and purified by various conventionally known methods, but it is the same as the method for purifying natural fish-derived transglutaminase. The recombinant transglutaminase can also be purified by the method described above. In addition, there is no report that sugar chains are added to natural fish-derived transglutaminase, but sugar chains are not added to recombinant transglutaminase produced using E. coli as a host, while yeast is used as a host. In such a case, addition of a sugar chain may be considered depending on the glycosylation function in the yeast cell.

The present invention will be described in more detail below with reference to examples.

[0026]

【Example】

[1. DNA fragment having a gene encoding red sea bream transglutaminase] 1.3 g of red sea bream liver 4M
It was crushed in a solution of guanidine thiocyanate, 1% β-mercaptoethanol (20 ml) using Polytron and a Teflon homogenizer. Chromosomal DNA was shredded by adding 0.5% sodium lauryl sarcosinate to the cell suspension to dissolve it, and then passing the solution through a 23-gauge injection needle 10 times. Next, this solution was centrifuged at 4 ° C. and 5000 rpm for 20 minutes, and the supernatant was collected. Further, according to a conventional method, the total RNA was purified from the supernatant by centrifugation with a density gradient of CsCl (Sambrook e
t al., Molecular Cloning: a laboratory manual, Cold
Spring Harbor Laboratory, Cold Spring Harbor Pres
s (1989)). The amount of total RNA obtained was 3.8 mg. 1.3 mg of this was applied to an mRNA purification kit (Clontech) using an oligo (dT) -cellulose column to purify the mRNA molecule to obtain about 20 μg.

Of the obtained mRNA, 8 μg of cDNA
Used as a template for fabrication. Random primers are used for cDNA synthesis, and double-stranded cDNA is synthesized Y
ou-prime cDNA synthesis kit (Pharma
cia) was used. The obtained cDNA was inserted into the restriction enzyme cleavage site EcoRI of the λ phage vector λZapII (Stratagene), and then the GIGAPACKII.
Using a packaging kit of GOLD (Stratagene), it was prepared and obtained as a cDNA library of red sea bream in a form incorporated into a phage protein. The titer of this library is 1.2X10.
6 pfu / μg vector.

From the above red sea bream cDNA library, a phage corresponding to 6.0 × 10 4 pfu was transformed into a host cell X.
After infection with L1-Blue, four agar plates with a diameter of 150 mm were spread on the plate at 1.5 × 10 4 pfu per plate. After culturing this at 37 ° C. for about 9.5 hours, the phage plaques formed on the plate were subjected to nylon membrane (Hibond-Amersham-made).
N). Next, the transferred nylon membrane was treated with alkali to denature the DNA, neutralized and washed. Then, the membrane was treated at 80 ° C. for 3 hours to immobilize the DNA on the membrane.

42 for the nylon membrane obtained
Pre-hybridization was performed at 2 ° C. for 2 hours, and then at 42 ° C. for 16 hours. The composition of the prehybridization solution was 6 × SSC (1 × SSC was 0.15M NaCl, 0.1%.
015M Sodium citrate, pH 7.0, 5X Denhardt's solution (1X
Denhardt's solution composition is 0.02% BSA, 0.02% Ficoll,
0.02% polyvinylpyrrolidone), 20% formamide, 100 μg / ml herring testis DNA, 0.1% SD
It consists of S. Also, during hybridization, DN
As an A probe, a synthetic DNA fragment (5'-GTCAA) near the active center of guinea pig transglutaminase was used.
GTACGGCCAGTGCTGGGTCTTCGC-
3 '; Ikura et al., Biochemistry 27, 2898-2905 (1
988): SEQ ID NO: 10) end-labeled with [γ- 32 P] ATP was used. The candidate strains of positive clones obtained by this screening were further subjected to secondary and tertiary screening to finally obtain 4 positive clones.

By infecting the infected cells carrying the above four positive clones with helper phage (R408), the cDNA derived from each positive clone was converted into a form incorporated in the phagemid vector pBluescriptSK-. The length of the inserted cDNA of each of the 4 clones is about 0.5 kbp (kilobase pairs), 1.5 kb.
p, 2.5 kbp and 1.0 kbp. And they were named pSLTG2, pSLTG4, pSLTG5, and pSLTG6, respectively.

Next, a restriction enzyme map of each cDNA clone was prepared, and Southern blotting analysis was performed using the inserted cDNA of pSLTG5 as a probe. As a result, pSLT was obtained.
G5 (with an insert cDNA length of 2.5 kbp) is the other 3
Since it was revealed that the cDNA clone contained the individual cDNA clones, the DN of the inserted cDNA of pSLTG5 was
The A base sequence was determined. The base sequence was analyzed by a conventionally known method using a Sequenase version 2.0 (U.S.B.) kit.

As a result, 2 shown in SEQ ID NO: 5 in the sequence listing
A DNA sequence containing a 520 base pair sequence was revealed. The restriction enzyme map of pSLTG5 is shown in FIG. In this sequence, there was a portion showing extremely high homology with the DNA probe used by the present inventors. The amino acid sequence translated from this base sequence is shown in SEQ ID NO: 2 in the sequence listing. In this amino acid sequence, Tyr-Gly-Gln-Cys-Trp-Val-Phe-, the active center sequence of 8 amino acid residues commonly present in guinea pig liver-derived transglutaminase and human blood coagulation factor XIII. Ala (SEQ ID NO: 1
1) [Nakanishi et al., Eur. J. Biochem. 202, 15-21
(1991)] existed. DNA containing the transglutaminase cDNA of red sea bream obtained as described above
E. coli strain carrying the plasmid pSLTG5 (AJ1267
3), Escherichia coli XL1-Blue / pSLTG5 has been deposited as Microindustrial Research Institute No. 4114 (FERM BP-4114).

[2. DNA fragment having a gene encoding transglutaminase of Alaska pollack] 2.3 g of liver of Alaska pollack, 4M guanidine thiocyanate,
The cells were disrupted using a Polytron and a Teflon homogenizer in a solution of 1% β-mercaptoethanol (20 ml). To this cell suspension was added 0.5% sodium lauryl sarcosinate to dissolve and then this solution was added to
Chromosomal DNA was shredded by passing it through a 3 gauge injection needle 10 times. Next, this solution is subjected to 10,000 rp at 4 ° C.
Centrifugation was performed for 20 minutes, and the supernatant was collected. Further, according to a conventional method, total RNA was purified from the supernatant by centrifugation with a density gradient of CsCl (Sambrook et al., Molecular Cloni
ng: a laboratory manual, Cold Spring Harbor Labora
See tory, Cold Spring Harbor Press (1989)). The amount of total RNA obtained was 7.2 mg. 2.3 of this
mg as mR using oligo (dT) -cellulose column
By applying the NA purification kit (Clontech), the mRNA molecule was purified to obtain about 23 μg.

Of the resulting mRNA, 4 μg of cDNA
Used as a template for fabrication. Random primers are used for cDNA synthesis, and double-stranded cDNA is synthesized Y
ou-prime cDNA synthesis kit (Pharma
cia) was used. The obtained cDNA was inserted into the restriction enzyme cleavage site EcoRI of the λ phage vector λZapII (Stratagene), and then the GIGAPACKII.
Using a packaging kit of GOLD (Stratagene), a cDNA library of Alaska pollack was prepared and obtained in a form incorporated into a phage protein. The titer of this library is 4.
1 × 10 5 pfu / μg vector.

After infecting a host cell XL1-Blue with a phage corresponding to 5.8 × 10 4 pfu from the above Alaska pollack cDNA library, 1.5 × 10 4 pf per plate was placed on 4 agar plates with a diameter of 150 mm.
sprinkled to be u. After culturing this at 37 ° C. for about 9.5 hours, the phage plaque formed on the plate was subjected to nylon membrane (Amersham's Hibond).
-N). Next, the transferred nylon membrane was treated with alkali to denature the DNA, neutralized and washed. Then, the membrane was treated at 80 ° C. for 3 hours to immobilize the DNA on the membrane.

42 for the nylon membrane obtained
Pre-hybridization was performed at 2 ° C. for 2 hours, and then at 42 ° C. for 16 hours. The composition of the prehybridization solution was 6 × SSC (1 × SSC was 0.15M NaCl, 0.1%.
015M Sodium citrate, pH 7.0, 5X Denhardt's solution (1X
Denhardt's solution composition is 0.02% BSA, 0.02% Ficoll,
0.02% polyvinylpyrrolidone), 20% formamide, 100 μg / ml herring testis DNA, 0.1% SD
It consists of S. Also, during hybridization, DN
As the A probe, the restriction enzyme C in the cDNA of red sea bream transglutaminase described in SEQ ID NO: 4 in the Sequence Listing is used.
Approximately 300 base pairs of D that can be cut out with laI and BamHI
An NA fragment (DNA capable of encoding a region containing an amino acid sequence near the active center) random-labeled with [α- 32 P] dCTP was used. The candidate strains of positive clones obtained by this screening were further subjected to secondary and tertiary screening to finally obtain 8 positive clones.

By infecting the infected cells carrying the above eight positive clones with helper phage (R408), the cDNA derived from each positive clone was converted into a form incorporated into the phagemid vector pBluescriptSK-. The name of each 8 clone is p
ALTG1, pALTG2, pALTG3, pALTG
6, pALTG7, pALTG8, pALTG9, pA
It was LTG10. Of these, pALTG1, 3, 6,
8, the length of the inserted cDNA was assayed, a restriction enzyme map was prepared, and the cDNA base sequences at the 5 ′ and 3 ′ ends were analyzed to obtain the correlation of each clone shown in FIG. . At this time, a fluorescent primer cycle sequencing kit (ABI) was used for the analysis of the base sequence.
(Manufactured by the company) was used.

Next, a synthetic primer (20 bases) was prepared based on the partial base sequence of the obtained cDNA, and pALTG8
The entire base sequence of the inserted cDNA was determined. Sequencing version 2.0 (U.S.B.
Company) kit was used. As a result, a DN of 2921 base pairs shown in SEQ ID NO: 9 in the sequence listing
The A sequence was revealed. The amino acid sequence translated from this cDNA base sequence is shown in SEQ ID NO: 6 in the sequence listing. In this amino acid sequence, a transglutaminase derived from guinea pig liver and an active center sequence of 8 amino acid residues commonly present in human blood coagulation factor XIII, T
yr-Gly-Gln-Cys-Trp-Val-Ph
There was e-Ala (SEQ ID NO: 11). Escherichia coli XLI-Blue / pA, an Escherichia coli strain (AJ 12709) carrying the plasmid pALTG8 containing the transglutaminase cDNA of Alaska pollack obtained as described above.
LTG8 has been deposited as Microorganism Research Institute No. 4115 (FERM BP-4115).

On the other hand, transglutaminase expressed in tissues other than liver of Alaska pollack was also examined. Therefore, transglutaminase c from muscle tissue
Attempts were made to clone the DNA.

11.5 g of Alaska pollack muscle was disrupted in a solution of 4 M guanidine thiocyanate, 1% β-mercaptoethanol (80 ml) using Polytron and a Teflon homogenizer. Chromosomal DNA was obtained by adding 0.5% sodium lauryl sarcosinate to the cell suspension to dissolve it, and then passing the solution through a 23-gauge needle 7 times and then a 25-gauge needle 7 times.
Shredded. Next, this solution is treated at 4 ° C. for 10,000 r
After centrifugation at pm for 20 minutes, the supernatant was collected. Further, according to a conventional method, the supernatant was subjected to a density gradient centrifugation operation of CsCl,
Total RNA was purified (Sambrook et al., Molecular Clo
ning: a laboratory manual, Cold Spring Harbor Labor
atory, Cold Spring Harbor Press (1989)). The amount of total RNA obtained was 2.1 mg. Of these 1.
7 mg was measured using an oligo (dT) -cellulose column.
The mRNA molecule was purified by applying an RNA purification kit (Clontech) to obtain about 21 μg.

3.2 μg of the obtained mRNA was cD
Used as a template for NA production. A random primer is used for cDNA synthesis, and a TimeSave cDNA synthesis kit (Pha
rmcia) was used.

Using this cDNA library, plaque hybridization was carried out by the experimental method as in the above-mentioned example, but a positive cDNA clone could not be obtained. Therefore, a cDNA fragment was obtained by the method described below.

Amplitaq DNA Poly was prepared by using the prepared Alaska pollack muscle cDNA group as a template and an oligonucleotide synthesized on the basis of the gene base sequence of Alaska pollack liver-derived transglutaminase as a primer.
A cDNA fragment of Alaska pollack muscle transglutaminase was specifically gene-amplified by the PCR method (polymerase chain reaction method) using merase (Takara Shuzo).

As shown in FIG. 10, the 5 ′ end region (N-terminal coding region) of the predicted transglutaminase cDNA structure derived from Alaska pollack muscle was used as a primer for synthesizing the cDNA fragment, Pr. Number 65 5'-TT
GGAAGCTTGTAAGAGCAACTCTTGGAAA-3 ') and Pr.970 (SEQ ID NO: 66 5'-TTGTACACTCGATCGATGGAGAGGT-3') were used. After PCR, a DNA fragment of about 980 bp could be amplified.
Next, the ends of this fragment were DNA Blunting K
After blunting with it (Takara Shuzo), it was incorporated into the restriction enzyme HincII cleavage site of pUC18 vector.

For gene amplification in the central region, Pr. 620 (SEQ ID NO: 675 ') was used as a synthetic oligonucleotide primer.
-TCTGCTTTGGGATCCTTGACCGCT-3 ') and Pr.2000 (SEQ ID NO: 685'-TGAAGGAGAGCTCCACAGACACA-3') were used. Among these primers are the restriction enzymes BamHI and S
The cleavage recognition site of acI was artificially incorporated. Therefore,
After PCR, an amplified DNA fragment of about 1.4 Kbp was prepared, and the digested DNA fragment was digested with the above restriction enzyme to obtain pB.
It was integrated into the same restriction enzyme site of luesscriptIISK- to obtain a cDNA clone.

In order to amplify the cDNA fragment of the 3'end region (C-terminal side coding region), first, PCR primer Pr.10 is used.
-1F (SEQ ID NO: 69 5'-ATGATGTCAAAGGCTGTCAC-3 ') and Pr.8-1R (SEQ ID NO: 70 5'-TCTTACCATATAAGTTGTAA-
The target region was amplified in 3 '). However, with these primers, amplification of small fragments other than the target DNA fragment was also observed. Therefore, using this amplified DNA group as a template, the same primer Pr.10-1F and a new primer, Pr.3-2F2R.
The gene was amplified again using (SEQ ID NO: 715'-ATTGATTAACAACAAAATGG-3 '). As a result, about 800 bp DNA
The fragment could be amplified. This cDNA fragment was also blunted at both ends as described above, and then pBluescriptIISK-
Was incorporated into the restriction enzyme EcoRV site.

Escherichia coli XL1-Blue was transformed with each of the plasmids holding the above-mentioned cDNA fragments having the three types of regions to obtain N. Clone Nos. N-3, N-4, and N-5 were designated as containing the central region. SB-4, SB-5, SB
-21, SB-22, and SB-30 are clones having the C-terminal side and are clone No. C-6, C-9,
C-13 was obtained. Next, the nucleotide sequences of the above 11 DNA clones were calculated using Taq D from Applied Biosystems.
yeDeoxy Terminator Cycle
Analysis was performed by a known method using the Sequencing Kit, and as a result, the DNA sequence shown in SEQ ID NO: 51 in the sequence listing and the same structure as the transglutaminase cDNA derived from Alaska pollack liver shown in SEQ ID NO: 8 were identified. And two DNA sequences having

From the above, the transglutaminase shown in SEQ ID NO: 6 is a pollack transglutaminase expressed in Alaska pollack, which is expressed across organ types.
The transglutaminase shown in SEQ ID NO: 49 could not be obtained because it was a full-length cDNA, but compared to liver-derived transglutaminase, a slight base substitution and 12 bp base deletion in the structural gene,
It was found that both genes and both gene products have extremely high homology, with only nucleotide insertions of 3 and 3 bp being observed.

Escherichia coli XLI, an Escherichia coli strain (AJ12790) carrying a plasmid N3 containing a part of the transglutaminase cDNA fragment derived from muscle of Alaska pollack (SEQ ID NO: 51) obtained as described above.
-Blue / N3 is Micromachine Research Institute, No. 4147 (FERM BP
-4147), and an Escherichia coli XLI-Blue / N5 Escherichia coli strain (AJ12791) carrying the plasmid N5.
Is a microscopic research institute No. 4148 (FERM BP-414
8), an E. coli strain carrying the plasmid SB4 (AJ127
92), Escherichia coli XLI-Blue / SB4 is Escherichia coli strain No. 4149 (FERM BP-4149), Escherichia coli strain harboring plasmid SB5 (AJ12793), Escher.
ichia coli XLI-Blue / SB5 is Escherichia coli X, which is an E. coli strain (AJ12794) harboring the plasmid SB21, which is Micromachine Research Institute No. 4150 (FERMBP-4150).
LI-Blue / SB21 is Micromachine Research Institute, No. 4151 (FERM
BP-4151), Escherichia coli XLI-Blue / SB, Escherichia coli strain carrying plasmid SB22 (AJ12795)
22 is Microtechnology Research Institute, No. 4152 (FERM BP-41
52) has been deposited.

[3. Construction of a plasmid pIL6TG1 expressing the red sea bream transglutaminase gene, introduction into Escherichia coli and assay of expression of physiological activity] Red sea bream transglutaminase gene (cDN prepared by the above-mentioned example)
As shown in FIG. 3, the plasmid pSLTG5 containing A) was digested with restriction enzymes XbaI and EcoRV to obtain a DNA fragment containing transglutaminase cDNA. On the other hand, the expression vector pBSF2-SD7 containing the tryptophan promoter and trpA terminator is Ba
After digestion with mHI and blunting of the DNA cleavage end with Klenow enzyme, treatment with XbaI was performed to obtain a large DNA fragment having a tryptophan promoter. In addition,
Expression plasmid pBSF2-SD7 is Bio / Technology 8
It is the plasmid described in p1036-p1040 (1990).

Two DNs obtained by processing as described above
The A fragment was ligated with T4 DNA ligase, and red sea bream transglutaminase cDNA expression plasmid pIL6T
G1 was obtained. When the DNA base sequence of the present pIL6TG1 was assayed, it was found that one base G of the BamHI cleavage site was deleted, and therefore an EcoRI site was present upstream of the trpA terminator in this plasmid. p
IL6TG1 was introduced into Escherichia coli HB101 by a known method to obtain a transformant, Escherichia coli HB101 / pIL6TG1,
(AJ12730) was produced. AJ12730 is a microbiological research
Deposited as No. 4116 (FERMBP-4116).

The obtained transformant colonies were spread on an agar plate containing 200 μg / ml of ampicillin,
After culturing at 30 ° C overnight, about 2c of cells on this plate
M2 casamino acid medium containing 200 μg / ml of ampicillin (Na2HPO4 · 12H2O 6g / l, KH2PO4 3g / l, NaCl 0.5g
/ l, NH4Cl 1g / l, casamino acid 2g / l, L-Leu 0.2g / l, L-Pro
0.2g / l, Thiamin ・ HCl 2mg / l, MgSO4 ・ 7H2O 0.5g / l, Ca
Cl2 · 2H2O 0.015 g / l, Glucose 2 g / l) 100 ml was inoculated into a Sakaguchi flask. This was cultured at 30 ° C. for about 16 hours to collect the bacterial cells.

0.5M EDTA solution 0.3m to the collected cells
30 ml of 20mM Tris-HCl, 30mM NaCl mixture was added,
Suspended. Further, 1 ml of 4 mg / ml lysozyme solution was added, and after stirring, the mixture was left at 0 ° C. for 1 hour. Then, the cell suspension was ultrasonically disrupted, and this was centrifuged (8000 rpm for 10 minutes) to prepare a cell disruption supernatant. Similarly, a centrifugal supernatant liquid of the disrupted bacterial cells was prepared from Escherichia coli (strain name E. coli HB101 / pBSF2-SD7) carrying a plasmid having no transglutaminase cDNA.

The transglutaminase activity of each supernatant was determined by measuring the fluorescence intensity due to the binding of monodansyl cadaverine to methylated casein (4 by the excitation wavelength light of 350 nm).
It was assayed by the activity detection method using the change in the fluorescence intensity at 80 nm) as an index. This activity detection method is based on Nippon Suisan Gakkaishi
(1991) Volume 57, pages 1203-1210, with minor modifications. That is,
Methylated casein 1 mg / ml, monodansyl cadaverine 15
150 μl of each assay sample was added to a solution (prepared to be 2.5 ml after sample addition) consisting of μM, CaCl2 5 mM, Tris-HCl (pH 7.5) 50 mM, DTT 3 mM, and after stirring, 37
The temperature was kept at 30 ° C. for 30 minutes. After the reaction, add EDTA solution to a final concentration of 10 mM.
The fluorescence intensity of each reaction solution was measured with a fluorescence intensity meter (Shimadzu RF-520).

[0055]

[Table 1]

As a result, as shown in Table 1, it was revealed that the cell extract of Escherichia coli carrying the expression plasmid containing the transglutaminase cDNA obtained in the above example clearly had transglutaminase activity. did. From this, it became clear that the cDNA obtained by us could encode transglutaminase.

[4. Construction of plasmid pTTG2-22 expressing red sea bream transglutaminase gene, introduction into Escherichia coli and assay of physiological activity expression] Next, in order to obtain more transglutaminase, it was decided to improve the transglutaminase expression plasmid. . This will be described below.

Since the expression level of transglutaminase by the transglutaminase expression plasmid pIL6TG1 obtained in Example 3 was low, pIL6TG was further added.
In order to construct a plasmid capable of expressing more than 1 transglutaminase, intensive research was conducted, and it was decided to devise to improve the translation efficiency of this transglutaminase gene.

The idea is to replace a part of the base sequence of the natural transglutaminase gene with a chemically synthesized DNA, modify the base sequence without changing the encoded amino acid sequence, and improve the efficiency of the present transglutaminase gene in E. coli. It was designed to be well expressed.
That is, as shown in FIG. 4, the region encoding the amino-terminal methionine of the present transglutaminase to the leucine at the 32nd position (selecting codons preferred in E. coli) and its consensus SD (Shine)
-Dalgarno) sequence (5'-TAAGGAGGT-
3 ') chemically synthesized DNA designed to exist
Fragments were prepared (SEQ ID NOS: 12 to 19 in Sequence Listing), and
This was done by the following procedure with the planned integration into the native transglutaminase gene.

Eight DNA oligomers constituting the chemically synthesized DNA-1 shown in FIG. 4 (SEQ ID NOS: 12 to 19 in Sequence Listing)
Was produced by a synthetic DNA device (manufactured by ABI). These oligomers were combined and ligated by an ordinary known method, that is, annealing and T4 DNA ligase to prepare chemically synthesized DNA-1. Next, the restriction enzyme EcoR
To pUC19 digested with I and HindIII, the DN
The A fragment was cloned to construct pFTGN6 (Fig. 5). In addition, using this plasmid, a DNA nucleotide sequence assay was performed, and it was confirmed that the prepared DNA had the intended sequence.

The plasmid pFTGN6 was digested with the restriction enzyme Cla.
I, HindIII digestion, and the restriction enzyme H
Treatment with aeII gave a DNA fragment C (about 110 base pairs) having ClaI and HaeII ends (FIG. 6).

On the other hand, a plasmid pSLTG5 containing cDNA encoding red sea bream transglutaminase was Ec
Approximately 1.44 kb with a portion of transglutaminase cDNA treated with oRI, HaeII and NcoI
A p (kilobase pair) DNA fragment (DNA fragment B) was obtained (FIG. 6).

Tryptophan promoter and trpA
Expression vector pT13sNco with terminator
(This plasmid is described in J. Biochem. 104, 30-34) was digested with NcoI and BamHI, each cut end was blunted with Klenow enzyme, and then a large DNA fragment was obtained. This was further self-ligated with T4 DNA ligase to obtain pTTNco. Next, this pTTNco was digested with ClaI and NcoI, and the digested end was dephosphorylated with alkaline phosphatase to prepare a large DNA fragment (DNA fragment A) having a tryptophan promoter and a terminator (FIG. 6).

Each of the DNA fragments A, B and C was replaced with T4 DNA.
By ligation with ligase, plasmid pTTG1
Was constructed (Fig. 7). In order to facilitate the subsequent understanding in FIGS. 7 and 8, the DN displayed by the “white bar” in FIG.
Fragment A is displayed as "black bar".

Next, the constructed plasmid pTTG1 was added to B
After treatment with amHI and blunting the cut ends with Klenow enzyme, digestion with SacI gave the large DNA fragment shown in FIG. Also, pSLTG5 was treated with restriction enzymes SacI and EcoRV to prepare small DNA fragments. The expression plasmid pTTG2-22 was constructed by ligating these DNA fragments with T4 DNA ligase (FIG. 8). pTTG2-22 was introduced into Escherichia coli HB101 by a known method to obtain a transformant Escherichia coli HB.
101 / pTTG2-22, (AJ12742) was prepared. AJ1274
2 has been deposited as Microindustrial Research Institute No. 4117 (FERM BP-4117).

The obtained transformant colony was spread on an agar plate containing 200 μg / ml of ampicillin,
After culturing at 30 ℃ overnight, the cells on this plate are about 2 cm.
2 to the M 9 casamino acid medium of 3 above (ampicillin 200
Sakaguchi flask containing 100 ml (containing μg / ml) was inoculated. This was cultured at 30 ° C. for about 16 hours to collect the bacterial cells.

From the collected cells, a cell disruption supernatant was prepared in the same manner as in the above 3. Similarly, a centrifugal supernatant liquid of the disrupted bacterial cells was prepared from E. coli (E. coli HB101 / pTTNco) carrying a plasmid having no transglutaminase cDNA.

The transglutaminase activity of each supernatant (50 μl) was set in the same manner as in the above 3, and the fluorescence intensity (35%) due to the binding of monodansyl cadaverine to methylated casein.
It was assayed by the activity detection method using the change in the fluorescence intensity of 480 nm by the excitation wavelength light of 0 nm as an index (Table 2). As a result, the transglutaminase c obtained in the above example was obtained.
It was revealed that a transglutaminase activity was clearly present in the Escherichia coli cell extract containing the expression plasmid (pTTG2-22) containing DNA. This further revealed that the cDNA we obtained could encode transglutaminase.

[0069]

[Table 2]

Next, it was tested whether the above-mentioned bacterial cell extract could gel the myosin B solution derived from Alaska pollack. Alaska pollack myosin B as a substrate (about 6 mg / ml)
To 1.0 ml of the solution, 471 μl of 50 mM CaCl2 was added, or under non-added conditions, 100 μl of the E. coli crude extract in which the transglutaminase activity was detected was added, or a non-added standard was prepared, and after stirring, It was left at room temperature for about 16 hours.

The gelation of myosin B was determined by inverting the test tube, which was the container in which the reaction was performed, and dropping the reaction contents or solidifying them. As a result, as shown in Table 3, in the presence of calcium chloride, gelation was observed only in the preparation containing the Escherichia coli extract that showed transglutaminase activity, and the cDNA obtained by the present invention does not contain transglutaminase. It was found to be the encoding gene.

[0072]

[Table 3]

[5. Construction of plasmid pYSTG 1 expressing red sea bream transglutaminase gene, introduction into baker's yeast Saccharomyces cerevisiae and assay of expression of physiological activity] Plasmid pSLTG5 containing cDNA encoding red sea bream transglutaminase is a restriction enzyme Ec
DN of about 2.5 kbp (kilo base pair) having cDNA of transglutaminase treated with oRI and XbaI
A fragment was obtained.

On the other hand, an expression vector in baker's yeast is GA, which is a gene encoding galactokinase.
P that can utilize the L1 promoter for expression of foreign genes
YES2 (INVITROGEN) was used. This was treated with the same restriction enzymes EcoRI and XbaI to give about 5.8k
A bp DNA fragment was prepared.

The above two DNA fragments were treated with T by a conventional method.
Ligation reaction was performed with 4DNA ligase, and this was transformed into competent cells of E. coli HB101 (manufactured by Takara Shuzo) according to a conventional method. For selection of transformants, L-agar plates containing the antibiotic ampicillin at a concentration of 100 μg / ml were used. Next, pick up 6 strains from the colonies of E. coli that have grown there with a sterilized toothpick,
The cells were inoculated into 3 ml of L-medium containing 100 μg / ml of ampicillin, and cultured with shaking at 30 ° C. for 16 hours.

Plasmid DNA was prepared from 2 ml of each cultured cell according to the alkaline SDS method, and the digestion pattern with restriction enzymes was analyzed by agarose gel electrophoresis. The plasmids obtained from the 4 strains were found to be the desired plasmids. Was identified and was named pYSTG1. Furthermore, the DNA base sequence of the ligated portion in the construction of this plasmid was analyzed to confirm that the base sequences were correct.

The pYSTG1 constructed as described above was subjected to the alkali cation method (purchased from Funakoshi Co., Ltd., Bio101).
Yeast Saccharomyces according to the yeast transformation method by
cerevisiae INVSC2 strain (MATα, his3-Δ200, ura3-16
It was introduced in 7). The histidine 20
YNB medium containing mg / l (composition 0.67% Yeast Nitrogen
Base w / o Amino acids (manufactured by Difco), 2% glucose, 2% agar) were used. Thus transformants, Saccharomyces ce
Obtained revisiae INVSC2 / pYSTG1, (AJ14679). In addition, AJ14679 is Microbiology Research Institute, No. BP-4085 (FERM BP-408
5) has been deposited as

The transformant yeast grown on the YNB medium plate containing 20 mg / l of histidine described above was inoculated into 100 ml of YPD medium (composition: 1% yeast extract, 2% bactopeptone, 2% glucose), and the temperature was 30 ° C. It was shaken and cultured overnight. Then, the cells were collected by centrifugation, washed with 1% yeast extract, and then suspended in 10 ml of the same solution. Next, 5%, which is half of this suspension, was mixed with 1% yeast extract and 2%.
% Bactopeptone and 2% galactose were added to 95 ml of a medium, and the mixture was shake-cultured at 30 ° C. for 18 hours. Transcription from the GAL1 promoter was induced by culturing the recombinant yeast in a galactose-containing medium. And the remaining 5 ml of suspension is 95 ml of liquid YPD medium.
Were similarly inoculated with and cultured under the condition that transcription from the GAL1 promoter was not induced.

On the other hand, the plasmid-free host yeast INVS
The C2 strain was also grown on a YPD medium plate, similarly inoculated into 100 ml of liquid YPD medium, and cultured at 30 ° C.
Subsequently, the above operation was carried out, and 5 ml of the obtained suspension was inoculated into 95 ml of liquid YPD medium and cultured.

The sample used for the measurement of transglutaminase activity was prepared as follows. The yeast cultivated in 100 ml of the medium was collected by centrifugation, and then 20 mM Tris-hydrochloric acid buffer containing 30 mM sodium chloride (pH 7.
15 ml of 5) was added to suspend the cells. Where 0.5
After adding 0.25 ml of M EDTA and stirring, glass beads (diameter 0.75 mm) of approximately equal volume were added to this suspension, and the mixture was vigorously stirred for 4 minutes. After that, 6000 rp
Insoluble matter was removed by centrifugation at m for 10 minutes, and the supernatant was used as a sample for transglutaminase activity measurement.

The transglutaminase activity of the supernatant (50 μl or 150 μl) of the disrupted cells was determined in the same manner as in Example 3 above, by the fluorescence intensity due to the binding of monodansyl cadaverine to dimethylated casein (excitation wavelength light of 350 nm). The activity was assayed using the change in the fluorescence intensity of 480 nm wavelength light) as an index (Table 4). The fluorescence intensity value obtained when a disrupted cell of yeast INVSC2 strain without plasmid was added to this activity measurement solution was set to 0. As a result, the transglutaminase c obtained by the present invention
It was clarified that a transglutaminase activity was apparently present in the disrupted cell extract of yeast in which the expression plasmid (pYSTG1) containing DNA was retained and transcription of the GAL1 promoter was induced. In addition, when EDTA was added to the enzyme reaction solution in advance to remove calcium ions, naturally no transglutaminase activity was detected. From this, we further obtained the cDN
It was revealed that A encodes transglutaminase.

[0082]

[Table 4]

[6. DNA fragment having a gene encoding a flounder transglutaminase] Flounder liver (1.5 g) was disrupted using a polytron and a Teflon homogenizer in a solution (20 ml) of 4 M guanidine thiocyanate and 1% β-mercaptoethanol.
Chromosomal DNA was shredded by adding 0.5% sodium lauryl sarcosinate to the cell suspension and dissolving it, and then passing this solution through a 25-gauge injection needle 8 times. Next, this solution was centrifuged at 4 ° C. at 10,000 rpm for 20 minutes, and the supernatant was collected. Further, according to a conventional method, total RNA was purified from the supernatant by centrifugation with a density gradient of CsCl (Sambrook et al., Molecular Cloning: a laborator.
y manual, Cold Spring Harbor Laboratory, Cold Spri
ng Harbor Press (1989)). The amount of total RNA obtained was 4.7 mg. About 1.6 mg of this was applied to an mRNA purification kit (Clontech) using an oligo (dT) -cellulose column to purify the mRNA molecule to about 23
μg was obtained.

Of the obtained mRNA, 4.4 μg was added to cD
Used as a template for NA production. A random primer is used for cDNA synthesis, and a TimeSave cDNA synthesis kit (Pha
rmcia) was used. The obtained cDNA was inserted into the restriction enzyme cleavage site EcoRI of λ phage vector λZapII (Stratagene), and then GIGAPA.
Using a packaging kit of CKII GOLD (Stratagene), it was prepared and obtained as a flounder cDNA library in a form incorporated into a phage protein. The titer of this library is 2.
It was a 0 × 10 5 pfu / μg vector.

From the above flounder cDNA library, a phage corresponding to 2.2 × 10 5 pfu was transformed into host cell X.
After infection with L1-Blue, agar with a diameter of 150 mm
11 plates were seeded with 2 × 10 4 pfu per plate. After culturing this at 37 ° C. for about 6 hours, the phage plaque formed on the plate was nylon membrane (Amersham's Hibond-N).
Transferred to. Next, the transferred nylon membrane was treated with alkali to denature the DNA, neutralized and washed.
Then, the membrane was treated at 80 ° C. for 2.5 hours to immobilize the DNA on the membrane.

42 for the nylon membrane obtained
Pre-hybridization was performed at 2 ° C. for 2 hours, and then at 42 ° C. for 16 hours. The composition of the prehybridization solution was 6 × SSC (1 × SSC was 0.15M NaCl, 0.1%.
015M Sodium citrate, pH 7.0, 5X Denhardt's solution (1X
Denhardt's solution composition is 0.02% BSA, 0.02% Ficoll,
0.02% polyvinylpyrrolidone), 20% formamide, 100 μg / ml herring testis DNA, 0.1% SD
It consists of S. Also, during hybridization, DN
As the A probe, the restriction enzyme C in the cDNA of red sea bream transglutaminase described in SEQ ID NO: 4 in the Sequence Listing is used.
Approximately 300 base pairs of D that can be cut out with laI and BamHI
An NA fragment (DNA capable of encoding a region containing an amino acid sequence near the active center) random-labeled with [α- 32 P] dCTP was used. The candidate strains of positive clones obtained by this screening were further subjected to secondary and tertiary screening to finally obtain 10 positive clones.

By infecting the infected cells carrying the above 10 positive clones with helper phage (R408), the cDNA derived from each positive clone was converted into the form incorporated in the phagemid vector pBluescriptSK-. Next, of these clones,
5 clones (pFLTG10, 12, 16, 17, 21
The length of the inserted cDNA was assayed, and a restriction enzyme map was prepared, and 5'end and 3'end c
The DNA base sequence was analyzed. At this time, for the analysis of the nucleotide sequence, a fluorescent primer cycle sequencing kit (manufactured by ABI) was used. As a result, it was revealed that each of the above clones was a cDNA fragment encoding the central part of the transglutaminase gene.

Therefore, in order to obtain a clone capable of encoding the C-terminal side of transglutaminase, which was not obtained by the above-mentioned 5 clones, the above flounder cDNA library was screened again as follows.

As a DNA probe for screening,
This time, the restriction enzyme EcoRI from clone pFLTG21
DNA fragment of about 300 base pairs that can be excised with
A DNA fragment of about 500 base pairs which can be excised from TG17 by treatment with restriction enzymes SalI and PstI is [α-32P] d.
The one randomly labeled with CTP was used. Other conditions were as described above. As a result, a positive clone was obtained by this screening, and finally, the secondary,
Ten positive clones were obtained by the third screening.

Of the 10 positive clones described above, 4 cDNA clones were cloned into pBl vector.
It was converted into uescript SK- and each correlation of the inserted cDNA was analyzed. In addition, this 4 clone is pFLTG4
4, 51, 55, 63.

Furthermore, in order to obtain a clone capable of encoding the N-terminal side of transglutaminase, a synthetic DN prepared based on the nucleotide sequence of the inserted cDNA 5'end of pFLTG12
A primer (consisting of 19 bases, the sequence is 5'-A
CACTGCCGGTCCCATCGAA-3 ') was used to newly prepare a flounder cDNA library.
2 μg of the above-mentioned mRNA sample was used as a template for cDNA synthesis. The titer of the library obtained here was 1.5 × 10 4 pfu / μg vector.

Next, 6X from the above cDNA library
A phage corresponding to 10 3 pfu was used as a host E. coli XL1-
Blue was infected and screened by the method described above. However, as a probe for hybridization, a DNA of about 300 base pairs that can be cut out from the above-mentioned clone pFLTG21 with a restriction enzyme EcoRI.
From the fragment and pFLTG17, the restriction enzymes SalI and Ps
A DNA fragment of about 500 base pairs which could be cut out by treatment with tI was randomly labeled with [α- 32 P] dCTP. The positive clone candidate strain obtained by this screening was further subjected to secondary and tertiary screening to finally obtain one positive cDNA clone.

The vector of this clone was converted into pBluescript SK- by the method described above, and this clone was designated as pFLTG60. Next, the length of the inserted cDNA of this clone was assayed, a restriction enzyme map was prepared, and the 5'-end and 3'-end cDNA nucleotide sequences were analyzed.

10 c obtained and analyzed as described above
Among the DNA clones, pFLTG21, 55, 60, 6
Of the inserted cDNAs of 3 and pFLTG17, the nucleotide sequence from the restriction enzyme SalI cleavage site to the 3 ′ end was analyzed. As a result, the DNA sequences shown in SEQ ID NOs: 45 and 47 in the sequence listing became clear. The difference between these two base sequences is the difference at the 1854th base. However, this mutation does not change the encoded amino acid. The amino acid sequences translated from these base sequences are shown in SEQ ID NOs: 43 and 44 in the sequence listing. Furthermore, the interrelationship among these clones is shown in FIG.

The flounder-derived transglutaminase cDNA fragment (SEQ ID NO: 4) obtained as described above was used.
5) Escherichia coli XLI-Blue / pF, which holds Escherichia coli XLI-Blue / pF, which holds the plasmid pFLTG21 containing a part of
LTG21 is Microtechnology Research Institute, No. 4154 (FERM BP-4
154), and Escherichia coli XLI-Blue / pF, an Escherichia coli strain (AJ12799) harboring the plasmid pFLTG55.
LTG55 is Microbiology Research Institute No. 4155 (FERM BP-4
155), an E. coli strain harboring the plasmid pFLTG60 (A
J12800), Escherichia coli XLI-Blue / pFLTG60
Micromachine Research Institute, No. 4156 (FERM BP-415
6), an E. coli strain carrying the plasmid pFLTG63 (AJ1
2801), and Escherichia coli XLI-Blue / pFLTG63 is Microorganism Research Institute No. 4157 (FERM BP-4157),
Plasmid pFLTG17 A plasmid pFL having a cDNA clone fragment downstream from SalI restriction enzyme of cDNA fragment
Escherichia coli strain (AJ12797) carrying TG17S, Escheri
chia coli XLI-Blue / pFLTG17SalI
It has been deposited as No. 53 (FERM BP-4153). In addition, each flounder-derived transglutaminase cD
The plasmid containing the NA fragment is shown in FIG.

Each cDNA fragment obtained as described above is
It can be easily converted into a single DNA fragment completely containing the transglutaminase coding region by a known method.
For this purpose, for example, pFLTG17S was cleaved with a restriction enzyme PstI, and a large DNA fragment obtained was treated with pFLTG63 in the same manner as Ps.
C of transglutaminase obtained by digesting with tI
The DNA fragment encoding the terminal side is ligated with T4 DNA ligase to construct a plasmid (named pFLTG1-C) in which the transglutaminase coding region is linked.

On the other hand, a large DNA fragment obtained by cleaving pFLTG21 with restriction enzymes BglII and PstI and a DNA obtained by similarly digesting pFLTG60 with restriction enzymes BglII and PstI and encoding the N-terminal side of transglutaminase obtained. The fragment is ligated with T4 DNA ligase to construct a plasmid (named pFLTG1-N) in which the transglutaminase coding region is linked. Then, after digesting this with EcoRI, both ends of the vector DNA fragment were dephosphorylated with a dephosphorylating enzyme, and the transglutaminase cDNA fragment of pFLTG1-N was added thereto again, and ligated to obtain pFTG1-N.
CD in the direction opposite to that of the cDNA fragment on LTG1-N
A plasmid pFLTG2-N having the NA fragment inserted therein is constructed.

Next, the plasmid pFLTG2-N was digested with the restriction enzyme Sa.
A cDNA fragment encoding the N-terminal side of transglutaminase can be obtained by digesting with lI. This fragment was digested with restriction enzyme SalI to pFLTG1-C
By ligation, a cDNA fragment in which all coding regions of transglutaminase are linked can be constructed.

On the other hand, for the E. coli strains recBC and sbcA, two cDNAs having overlapping cDNA regions were used.
Fragments (eg pFLTG60 and pFLTG21, pFLTG21 and pFLTG1
7, cDNA fragments on the pFLTG17 and pFLTG63 plasmids) are transformed into single-stranded plasmid DNA at both ends, so that the duplication sites in the cDNA fragments can be extremely easily formed by the recombination mechanism in the host E. coli. Then, recombination occurs, and a continuous cDNA fragment is constructed. By repeating this, the desired full-length cDNA can be obtained. Many chimeric genes have been constructed using such a recombination function (Ogawa et al., Journal of Mo.
lecular Biology, Vol.226, 651-660, 1992).

The present inventors also found that transglutaminase cD derived from the above red sea bream, Alaska pollack, and Japanese flounder.
When NA was obtained and the translated amino acid sequence was analyzed, the presence of a common amino acid sequence among the amino acid sequences constituting fish transglutaminase was found. These are SEQ ID NOS: 53, 54, 55, 56, 5 in the sequence listing.
7, 58, 59, 60, 61, 62, 63, 64. However, amino acids that are not specific amino acid residues are indicated by Xaa.

[7. Examination of whether or not a gene homologous to the obtained red sea bream transglutaminase cDNA is present in many other fish species] The present inventors have found that the transglutaminase gene derived from red sea bream obtained as in the above Examples is very similar to We examined whether highly homologous genes are present in other fish species.

Samples to be examined include horse mackerel, yellowtail,
Sardines, skipjacks, saury, mackerel, salmon liver, puffer fish ovaries, and Bacillus subtilis chromosomes were used as control samples. After about 3 g of each fish tissue was finely cut, 30 ml of ice-cooled TN buffer (composition: 20 mM Tris-HCl buffer (pH 7.5) containing 0.1 M sodium chloride) was added and mashed with a Teflon homogenizer.

The suspended sample was put in a centrifuge tube and centrifuged at 5 ° C. and 1500 rpm for 5 minutes in a cooling centrifuge to obtain a precipitate fraction. To this, 5 ml of ice-cold TNE buffer solution (TN buffer solution having a composition of 1 mM EDTA) was added and well suspended, and then 15 ml of ice-cooled TNE buffer solution was further added and mixed. afterwards,
Add 1 ml of 10% SDS and shake at room temperature for 30 minutes, then
Add 100 μl of 20 mg / ml Protease K solution and
The reaction was carried out at 0 ° C overnight.

After the reaction, the aqueous solution of each sample was subjected to phenol treatment, phenol chloroform treatment, and chloroform treatment for deproteinization.
Then, 1/50 volume of 5M sodium chloride and 2.5 volumes of ethanol were added to each sample to precipitate and recover nucleic acid, and finally 1 ml of TE buffer solution (composition was 1 mM E
It was dissolved in 10 mM Tris-HCl buffer (pH 7.6) containing DTA.

Next, about 10 μg of DNA of each sample was added to 200
It was digested with the unit restriction enzymes HindIII or HincII and EcoRI, and this was electrophoresed on a 1% agarose gel. After staining the gel with ethidium bromide and confirming that the amount of electrophoretic DNA in each sample was almost constant, the gel was immersed in 0.25N hydrochloric acid, and then 0.4N sodium hydroxide solution containing 0.6M sodium chloride. Processed.
After that, the gel was neutralized with 0.5 M Tris-HCl buffer (pH 7.5) containing 1.5 M sodium chloride, and 10X SSC (composition:
1.5M sodium chloride, 0.15M trisodium citrate)
The nucleic acid in the gel with a nylon membrane (GeneScreen Pl
us).

The membrane with adsorbed nucleic acid was treated with 0.4 N sodium hydroxide containing 0.6 M sodium chloride followed by 1.
0.5M Tris-HCl buffer containing 5M sodium chloride (pH 7.
After the treatment in 5), it was immersed in a 2X SSC solution. Then, the membrane was left at room temperature for 30 minutes and then dried at 80 ° C. for 2 hours.

65 with respect to the obtained nylon membrane
Prehybridization was performed at 3 ° C for 3 hours, and then at 60 ° C for 16 hours. The composition of the prehybridization solution was 5 × SSC (1 × SSC was 0.15M NaCl, 0.1%.
015M Sodium citrate, pH 7.0, 1X Denhardt's solution (1X
Denhardt's solution composition is 0.02% BSA, 0.02% Ficoll,
0.02% polyvinylpyrrolidone) and 1% SDS. Also, the solution composition during hybridization is 0.
75M sodium chloride, 20mM Tris-HCl (pH8.0), 0.25mM E
These were DTA, 1% SDS, 1X Denhardt's solution, and 50 μg / ml E. coli genome solution. Then, D prepared as follows
NA probe was used at a concentration of 2 × 10 5 cpm / ml.

As the DNA probe, a guinea pig transglutaminase cDNA fragment and a red sea bream transglutaminase cDNA fragment obtained by the present inventors were used as templates and radiolabeled with random primers. The guinea pig transglutaminase cDNA fragment was obtained by using the plasmid pKTG1 (Ikura et al., Eur. J. Biochem, 187, 705-71) containing it.
1, described in 1990) is treated with a restriction enzyme StuI to obtain a transglutaminase cDNA fragment. In the case of red sea bream, the obtained cDNA clone
pSLTG5 was cleaved with restriction enzymes ApaI and SacI, and the resulting transglutaminase cDNA fragment was used as a template.

The membrane hybridized as described above was washed with a 0.1X SSC, 0.1% SDS solution at 60 ° C., dried and then autoradiographed. As a result, under the present experimental conditions, the DNA probe derived from guinea pig did not pair with the nucleic acid of all the experimental samples, but the DNA probe derived from red sea bream did not pair with the chromosomal DNA of Bacillus subtilis at all. But, horse mackerel,
It was found to be strongly paired with nucleic acids derived from yellowtail, mackerel, bonito, and puffer fish, and weakly paired with nucleic acids derived from sardines, saury, and salmon.

From the above facts, it was shown that a gene highly homologous to the transglutaminase gene obtained by the present inventors exists also in other fish species, and at the same time, the obtained transglutaminase was used as a DNA probe. Thus, it was possible for the first time to show that the transglutaminase gene can be obtained in fish species other than red sea bream, pollock, and flounder extremely easily.

[8. Partial amino acid sequence analysis of transglutaminase of Alaska pollack liver] A transglutaminase gene derived from Alaska pollack obtained as described above,
In order to show that the transglutaminase enzyme actually expressed can be expressed, this enzyme was purified and purified, and its structure was clarified. The transglutaminase activity was assayed based on the activity detection method described above, using the change in fluorescence intensity due to the binding of monodansyl cadaverine to dimethylated casein as an index.

[0112] 15 g of Alaska pollack liver was supplemented with 10 mM NaCl, 5 mM ED
30 ml of 20 mM Tris-HCl buffer (pH 8.3) containing TA and 2 mM dithiothreitol was added, and the mixture was homogenized with a homogenizer. The disrupted solution was centrifuged at 4 ° C for 10 minutes at 3,000 rpm (Hitachi Himac CR 20B2, rotor RPR20-2). Then, the supernatant is further centrifuged at 37,000 rpm for 1 hour at 4 ° C (Hitachi 70P-72, rotor RP-70T), and the supernatant is applied to a 0.45 µm filter (GL Science GL chromatography disk) to obtain 24 ml of crude extract. Got

The protein concentration of this solution was measured by a protein assay kit manufactured by BioRad, and was about 8.6 m.
It was g / ml. In addition, when the transglutaminase activity was examined using 5 μl of this crude extract, the total activity was 849 units, and therefore the specific activity was 4.10 unit / mg protein.

Next, a column packed with Q-sepharose (Pharmacia φ1.6 cm x 10c), which was obtained by equilibrating the crude extract with the same buffer, was used.
When the solution was passed through m), transglutaminase was adsorbed. An attempt was made to elute transglutaminase using a NaCl concentration gradient, and it was eluted at a NaCl concentration of about 100 mM.
The obtained transglutaminase activity fraction (10 ml) was dialyzed against the same buffer overnight, passed through a Q-Sepharose packed column again, and eluted under the same conditions to obtain a transglutaminase activity fraction (9.5 ml).

The protein concentration of this active fraction is about 0.73 mg /
It was ml. Also, the total activity of transglutaminase is
It was 249 units and the specific activity was 36.1 units / mg protein.

Next, the obtained active fraction was treated with 50 mM NaCl and 2 mM E.
A column packed with S-Sepharose (Pharmacia φ1.6 cm x 10c, which was dialyzed overnight against sodium acetate (pH 6.45) containing DTA, 0.5 mM dithiothreitol and equilibrated with the same buffer solution.
When the solution was passed through m), transglutaminase was adsorbed. Transglutaminase was eluted with a NaCl concentration gradient (6.0 ml) at a NaCl concentration of about 200 mM.

The protein concentration of this active fraction is about 56 μg / m 2.
It was l. The total transglutaminase activity is 20
1 unit and the specific activity was 591.2 units / mg protein.

As shown below, only a protein showing a single band having a molecular weight of about 77,000 was present in this fraction, and transglutaminase derived from Alaska pollack liver was purified and obtained. The specific activity of the obtained purified solution was 143 times that of the crude extract, and the recovery rate was 23.7%.

A) Electrophoresis analysis 30 μl of purified transglutaminase was taken and the same amount of 10% was added.
Mercaptoethanol, 4% SDS, 20% glycerin, 0.002%
0.125 M Tris-hydrochloric acid buffer solution (pH 6.8) containing bromphenol blue was added, and heated in a boiling bath for 1 minute to prepare an electrophoretic sample. 40 μl of the electrophoretic sample was applied to a 5-20% polyacrylamide preformed gel manufactured by Atto Co., Ltd., and electrophoresed with 0.025 M Trisglycine buffer containing 0.1% SDS at 40 mA for about 2 hours. After completion of the electrophoresis, the plate was stained overnight with a 0.12% Coomassie Brilliant Blue solution containing 50% methanol and 7% acetic acid, and decolorized with a 7% acetic acid solution containing 50% methanol. As a result, the molecular weight is about 7
A single band was obtained at 7,000 positions.

B) Partial amino acid sequence of transglutaminase derived from Alaska pollack liver About 4 ml of S-Sepharose fraction containing about 80 μg of purified transglutaminase was put into a dialysis tube, and 0.001 mM E was added.
6 against 5 mM Tris-HCl buffer (pH 8.3) containing DTA
After dialysis for an hour, the solution was dialyzed again for 12 hours to remove the alkali metal ion in the transglutaminase enzyme preparation. This is concentrated by centrifugation to dryness and distilled water 0.8m
1 was added, and the mixture was stirred at 37 ° C. for 30 minutes and redissolved. 16 μg of trypsin (11,700 units / mg manufactured by Sigma) was added to this and reacted at 37 ° C. for 12 hours to decompose into peptide fragments. The reaction was terminated by dropping one drop of formic acid.

Next, this reaction solution was subjected to reverse phase HPLC (Inertsil Pre
p-ODS φ6.0 mm x 250 mm GL Science) was used, the solvent was 0.05% TFA (trifluoroacetic acid), and each peptide fragment was separated and obtained under elution conditions with a concentration gradient of acetonitrile.

The obtained peptide fragment was subjected to a protein sequencer (6400/6600 manufactured by MilliGen Biosearch),
When the amino acid sequences thereof were analyzed, the following 6 sequences were obtained. That is, Xaa-Ala-Gly-Gly-Ser-Gly-As
p (SEQ ID NO: 20), Trp-Trp-Leu-His-Gln-Gln-Ser
(SEQ ID NO: 21), Met-Tyr-Leu-Leu-Phe-Asn-Pro (SEQ ID NO: 22), Trp-Gln-Glu-Pro-Tyr-Thr-Gly-Gly (SEQ ID NO: 23), Phe-Asp- Val-Pro-Phe-Val-Phe-Ala-Glu
-Val-Asn-Ala-Asp (SEQ ID NO: 24), Ser-Xaa-Tyr-Ser-
It was Asn-Glu (SEQ ID NO: 25). However, Xaa represents an unidentified amino acid residue.

The above six amino acid sequences are completely in agreement with a part of the amino acid sequences (SEQ ID NOs: 6 and 7) predicted from the nucleotide sequences of the cDNAs already obtained by the present inventors. It was shown that the obtained cDNA encodes a transglutaminase that is actually expressed in the living body of Alaska pollack and has activity.

[9. Partial amino acid sequence analysis of red sea bream transglutaminase] On the other hand, in order to show that the red sea bream-derived transglutaminase gene obtained as described above can actually express the transglutaminase enzyme expressed in red sea bream. , Purify this enzyme,
It was purified and the structure was clarified. In addition,
The transglutaminase activity was assayed based on the above-described activity detection method using the change in fluorescence intensity due to the binding of monodansyl cadaverine to dimethylated casein as an index, as in the case of Alaska pollack.

20 g of red sea bream liver was supplemented with 10 mM NaCl, 5 mM EDTA,
46 ml of 20 mM Tris-hydrochloric acid buffer (pH 8.3) containing 2 mM dithiothreitol was added, and the mixture was homogenized with a homogenizer. Put this disrupted liquid in a centrifuge tube and
Centrifuge for 45 minutes at 50,000 rpm at 70 ℃ (Hitachi 70P-72
Rotor RP-65T) was run, and the supernatant liquid portion was passed through a 0.45 μm filter (Advantech DISMIC-25 disposable syringe filter unit) to remove insoluble polymer. As a result, 30 ml of a red-colored extract was obtained. Next, this extract was ice-cooled with 5 mM Tris-HCl buffer.
(pH 8.3) was added in an approximately equal amount (30 ml) to reduce the ionic strength of the solution, which was used as a crude extract of red sea bream liver (60 ml).

The protein concentration of this solution was measured by a protein assay kit manufactured by BioRad, and was found to be about 8.4 m.
It was g / ml. In addition, when the transglutaminase activity was examined using 5 μl of this crude extract, the total activity was 2088 units, and therefore the specific activity was 4.14 units / mg protein.

Subsequently, this crude extract was treated with 5 mM NaCl and 2.5 mM E.
DEAE-Sephacel packed column equilibrated with 10 mM Tris-HCl buffer (pH 8.3) containing DTA 0.5 mM dithiothreitol
After passing through (Pharmacia φ2.6cm X 11cm),
It was found that transglutaminase was adsorbed on the ion exchanger. Therefore, when we tried to elute from this column with a salt concentration gradient of NaCl, at a NaCl concentration of about 100 mM,
Fractions with transglutaminase activity can be eluted,
This was designated as the DEAE fraction (about 59 ml).

The protein concentration of this active fraction is about 145 μg /
It was ml. Also, the total activity of transglutaminase is
It was 1045 units and the specific activity was 122 units / mg.

The transglutaminase active fraction fractionated by DEAE-Sephacel resin was then placed in dialysis tubing and 20 mM containing 2 mM EDTA, 0.5 mM dithiothreitol.
It was dialyzed overnight against a sodium acetate buffer (pH 6.25). Then, this sample was passed through a CM-sepharose packed column (Pharmacia φ1.6 cm x 10 cm) equilibrated with the same buffer, and transglutaminase was adsorbed to this column. Therefore, when we tried to elute the enzyme from this column using a NaCl salt concentration gradient, a transglutaminase activity fraction was eluted at a NaCl concentration of about 200 mM, and it was obtained (about 35 ml), which was used as the CM fraction. .

The protein concentration of this active fraction is about 20 μg / m 2.
It was l. The total transglutaminase activity is about
It was 530.6 units and the specific activity was 758 units / mg protein.

The transglutaminase activity fraction fractionated by the CM-Sepharose resin was again put into a dialysis tube, and was added to a 20 mM sodium acetate buffer solution (pH 6.45) containing 50 mM NaCl, 2 mM EDTA and 0.5 mM dithiothreitol. And dialyzed overnight to reduce the salt concentration of the transglutaminase solution. When this was passed through a heparin-sepharose packed column (Pharmacia Hi-Trap affinity column capacity 1 ml) equilibrated with the same buffer, transglutaminase was adsorbed on the same resin. By applying a NaCl concentration gradient, transglutaminase was eluted from the heparin column at a NaCl concentration of about 200 mM and obtained (about 12.5 ml). This was designated as a heparin fraction.

The protein concentration of this active fraction is about 32 μg / m 2.
It was l. The total transglutaminase activity is about
It was 290.5 units and the specific activity was 807 units / mg protein.

This heparin fraction was subjected to SDS-polyacrylamide gel electrophoresis, and after electrophoresis, the gel was stained with Coomassie Brilliant Blue.
A single band stained only at 7,000 positions could be confirmed. Here, transglutaminase derived from red sea bream liver could be purified and obtained. The specific activity of the obtained purified solution of red sea bream transglutaminase was about 195 times that of the crude extract, and the recovery rate was about 14%.

Therefore, the partial amino acid sequence of red sea bream liver-derived transglutaminase was analyzed. About 4 ml of heparin fraction containing about 100 μg of purified transglutaminase
Was placed in a dialysis tube and dialyzed for 13 hours against 20 mM Tris-HCl buffer (pH 8.3) containing 0.1 mM EDTA and 0.01 mM dithiothreitol, followed by 20 mM Tris-HCl buffer containing 0.001 mM EDTA. The solution (pH 8.3) was dialyzed again to remove the alkali metal ion in the transglutaminase enzyme preparation. After adding 480 mg of urea to this and treating at 37 ° C for 30 minutes, lysyl endopeptidase (Wako Pure Chemical Industries) 7.
5 μg (0.02 unit (amidase activity)) was added and further subjected to enzyme treatment at 37 ° C. for 12 hours to fragment transglutaminase into peptide fragments. After the treatment, 40 μl of 10% TFA (trifluoroacetic acid) solution was added to the reaction solution (final concentration 0.1%), and the mixture was stirred.

Next, this reaction solution was subjected to reverse-phase HPLC (Vydac C4
A column, φ 4.6 mm x 250 mm) was used, the solvent was 0.1% TFA, and each peptide fragment was separated and obtained under elution conditions with a concentration gradient of acetonitrile.

The obtained peptide fragments were subjected to a protein sequencer (470A manufactured by Applied Biosystems), and their amino acid sequences were analyzed by a sequence analyzer (120A manufactured by Applied Biosystems).
The following sequence was obtained. That is, His-His-Leu-Glu-Leu-Val-
Leu-Xaa-Leu-Gly (SEQ ID NO: 26), Xaa-Xaa-Phe-Asn-G
ln-Gln-Gly-Ala-Gln-Asp-Glu-Ile-Leu-Leu-Thr-Leu-His
(SEQ ID NO: 27), Ile-Ser-Phe-His-Met-Leu-Phe-Asn
-Pro (SEQ ID NO: 28), Leu-Gln-Glu-Tyr-Val-Met-Asn-
Glu-Asp-Gly-Val-Ile-Tyr-Met-Gly-Thr (SEQ ID NO: 2
9), Asn-Ser-Glu-Met-Asp-Ile-Glu-His-Arg-Ser-As
p-Pro-Val-Tyr-Val-Gly-Arg-Thr (SEQ ID NO: 30), Ty
r-Asp-Ala-Pro-Phe-Val-Phe-Ala-Glu-Val-Asn-Ala-Asp-
Thr-Ile-Tyr (SEQ ID NO: 31), Ser-Val-Tyr-Gly-Asn-H
is-Arg-Glu-Asp-Val-Thr-Leu-His-Tyr (SEQ ID NO: 3
2), Ala-Gly-Arg-Arg-Val-Thr-Glu-Pro-Ser-Asn-Glu-
Ile-Ala-Glu-Gln-Gly-Arg-Leu (SEQ ID NO: 33), Xaa-A
la-Gln-Pro-Val-Phe-Gly-Thr-Asp-Phe-Asp-Val-Ile-Val
-Glu (SEQ ID NO: 34), Asn-Glu-Gly-Gly-Arg-Asp-Al
a-His-Ala-Gln-Leu-Thr-Xaa-Leu-Ala-Xaa-Ala (SEQ ID NO: 35), Thr-Ile-Ser-Val-Thr-Val-Pro-Ala-His (SEQ ID NO: 36), Ala-Val-Val-Xaa-Glu-Pro-Leu-Thr-Ala
(SEQ ID NO: 37), Gly-Gly-Val-Phe-Thr-Leu-Glu-gly
-Ala-Gly-Leu-Leu-Ser-Ala-Thr-Gln-Ile-His (SEQ ID NO: 38), Leu-Ser-Phe-Ser-Pro-Met-Arg-Thr-Gly-Val-
Arg (SEQ ID NO: 39), Leu-Leu-Val-Asp-Phe-Asp-Ser
-Asp-Arg-Leu (SEQ ID NO: 40), Gly-Val-Thr-Thr-Val-
Val-Val-His (SEQ ID NO: 41), Tyr-Arg-Ser-Leu-Ile-T
It was hr-Gly-Leu-His-Thr-Asp (SEQ ID NO: 42).
However, Xaa represents an unidentified amino acid residue.

These amino acid sequences are present in the amino acid sequences (SEQ ID NOs: 2 and 3) considered from the cDNA sequences obtained in the above examples, and this is true in the living body of red sea bream. It is shown that the obtained cDNA encodes a functioning enzyme showing the activity of transglutaminase.

[10. Comparison of Enzymatic Properties of Guinea Pig-Derived Transglutaminase and Fish-Derived Transglutaminase] The enzymatic properties of fish-derived transglutaminase (hereinafter abbreviated as FTG) were tested in comparison with guinea pig transglutaminase (hereinafter abbreviated as MTG) to obtain FT.
We examined the superiority of G in industrial use.

A) Specific activity In the above Examples, the enzyme specific activities of the purified and purified Alaska pollack transglutaminase and the commercially available MTG were compared and examined when the fluorescence method was used. MTG
Used were those manufactured by Takara Shuzo Co., Ltd. and those manufactured by Sigma. The MTG manufactured by Takara Shuzo had a purity of 95%, and the MTG manufactured by Sigma Co. had the same purity.

The above three transglutaminase preparations were prepared so as to have equivalent enzyme concentrations (determined by protein concentration measurement using a protein assay kit manufactured by BioRad),
When the transglutaminase activity was measured at pH 8.5 near the optimum pH for each, it was found that FTG exhibited 10 times the specific activity of MTG (manufactured by Takara Shuzo) and 20 times the specific activity of MTG manufactured by Sigma.

The transglutaminase preparations prepared at the three concentrations described above were developed by SDS-PAGE, stained with Coomassie Brilliant Blue, and subjected to densitometer (Ultra Scan XL Laser, LKB).
r Densitometer), the amount of each transglutaminase was assayed, and it was shown that FTG had a concentration about 4 times that of MTG. Considering this fact, it was found that the specific transglutaminase activity of FTG by the fluorescence method is 2.5 to 5 times that of MTG.

As described above, as compared with the specific activity of MTG, F
The high specific activity property of TG is considered to contribute to the cost reduction of product manufacturing because it reduces the amount of transglutaminase used for the expression amount of the same effect in its industrial application.

B) Thermal Stability Next, the ease of thermal deactivation was compared and examined between FTG and MTG. The amount of enzyme used was an amount capable of showing the same activity. First, prepare each transglutaminase preparation in a Tris-HCl buffer (pH 8.5) (composition: 250 μl of 0.5 M Tris-HCl buffer (pH 8.5), 80 μl of 100 mM DTT, 37 μl of 1 mM monodansyl cadaverine, and 1300 water). ), 0 ° C, or 20 ° C, 25 ° C, 30 ° C, 37 ° C, 4
The treatment was carried out at 0 ° C., 42 ° C., 50 ° C. and 60 ° C. for 10 minutes, then ice-cooled for 3 minutes, and each temperature treatment was stopped. Then there 250 μl of 10 mg / ml dimethylated casein solution, and 5
500 μl of 0 mM calcium chloride solution was added, and the mixture was reacted at 37 ° C. for 60 minutes. Then, add 0.5M EDTA to each reaction mixture.
100 μl was added to stop the enzyme reaction.

The fluorescence intensity of the reaction solution prepared as described above was measured to examine the thermal stability of each enzyme. as a result,
As shown in FIG. 13, it was revealed that the treatment temperature for giving 50% residual activity was about 9 ° C. lower in FTG than in MTG. This indicates that FTG is easier to inactivate than MTG. Combined with the result of a) above, FTG is more likely to undergo an enzymatic reaction, and the reaction product is inactivated by a slight heat treatment. It can be said that

C) Reactivity to actomyosin The preparation of actomyosin (AM) was carried out as follows. First of all, frozen surimi of Alaska pollack (Taiyo Fisheries Co., Ltd.)
SA grade) (about 30 g) and about 60 g of 20 mM Tris-HCl buffer solution (pH 7.5) containing 0.5 M sodium chloride.
After adding ml, the mixture was homogenized with an Excel auto homogenizer (manufactured by Nissei Sangyo Co., Ltd.). 1000 this next
Centrifuge 3 times at 0 rpm for 30 seconds (Hitachi HimacC
The insoluble matter was removed using a R20B2 centrifuge and rotor RPR20-2). Add the supernatant solution to a dialysis tube (S
eamess cellulose tubing,
The import source is Sanko Junyaku Co., Ltd., and this is a 20 mM Tris-HCl buffer containing 0.5 M sodium chloride (pH 7.
For 5), it was dialyzed for 3 hours and for 16 hours.

Then, the dialyzed solution was placed at 14000 rpm.
After centrifuging at 60 minutes and collecting the supernatant, it was filtered with gauze to prepare an actomyosin solution (about 27 ml). The protein concentration of this solution was measured with a protein assay kit manufactured by BioRad.
It was 1 mg / ml.

Using actomyosin prepared as described above, the effects of MTG and F on the polymerization of myosin protein were investigated.
The effects of TG were compared. In addition, polymerization of myosin,
A large amount of "Kamaboko" has the elasticity exerting function of fish paste products.
It is an index closely related to "deliciousness" (Numakura et al., Journal of the Fisheries Society of Japan, Vol. 51, No. 9, 1559-1565, (198
5), Seki et al., Journal of Japan Fisheries Society Vol. 56, No. 1, 125-132,
(1990)).

The actomyosin prepared as described above was adjusted to a concentration of 10 mg / ml with a 20 mM Tris-hydrochloric acid buffer solution (pH 7.5) containing 0.5 M sodium chloride, and the test tube (made by Terumo, washed) Test tube Larvo, 1
5.5 × 100 mm), 1 ml each was prepared. 5 to this
After adding 200 μl of 0 mM calcium chloride and stirring,
200 μl of separately prepared FTG or MTG (same active component in fluorescence activity measurement) was added and sufficiently stirred.
The MTG used was manufactured by Sigma.

After addition of each transglutaminase, 37
The reaction was carried out for 15 minutes, 30 minutes, 45 minutes, and 60 minutes at ℃. After each reaction, 100 μl of the reaction solution was withdrawn from the test tube, and 20 mM Tris-hydrochloric acid containing 8 M urea, 2% SDS, and 2% β-mercaptoethanol (pH 8.
The reaction was stopped by adding 100 μl of each of 5). Next, 4% SDS, 10% β-mercaptoethanol, 2
125 μm Tris-HCl buffer (pH 6.8) containing 0% glycerin and 0.002% bromophenol blue was further added thereto in an amount of 300 μl each, and these samples were heated at 100 ° C. for 1 minute, and then used for SDS-PAGE. Sample.

5 μl of each sample above was subjected to SDS-PAG
E, subjected to electrophoresis, and then stained with Coomassie Brilliant Blue to measure the amount of myosin H chain monomer in a densitometer (L
Ultro scan XL Laser manufactured by KB
Densitometer) to analyze the effect of each transglutaminase on the polymerization and multimerization of myosin protein. The result is shown in FIG.
It was found that G polymerizes myosin protein faster than MTG. This further suggests that in the application of transglutaminase to fish paste products, FTG is more likely to contribute to manufacturing cost reduction than MTG.

[0151]

The source of transglutaminase that can be used in the past has been actinomycetes and guinea pigs. However, according to the present invention, it is traditionally possible to retain or reinforce texture during processing of seafood such as kamaboko. Express function,
It has become possible to provide a DNA fragment having a fish-derived transglutaminase gene whose action in natural products is known. Mass production of transglutaminase is possible by applying gene recombination technology to the gene of fish transglutaminase obtained by the present invention, and by applying the produced enzyme, modification of physical properties of food protein and improvement of nutritional value Is possible. In addition to foods, the enzyme can be expected to be applied to pharmaceuticals and chemical products.

[0152]

[Sequence list]

SEQ ID NO: 1 Sequence Length: 25 Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Origin Organism Name: Sea Bream Liver Sequence Val Lys Tyr Gly Gln Cys Trp Val Phe Ala Ala Val Ala Cys Thr 1 5 10 15 Val Leu Arg Cys Leu Gly Ile Pro Thr Arg 16 20 25 SEQ ID NO: 2 Sequence Length: 694 Sequence Type: Amino Acid Topology: Linear Sequence Type: Protein Origin Organism Name: Sea Bream Liver Sequence Ala Ser Tyr Lys Gly Leu Ile Val Asp Val Asn Gly Arg Ser His 1 5 10 15 Glu Asn Asn Leu Ala His Arg Thr Arg Glu Ile Asp Arg Glu Arg 20 25 30 Leu Ile Val Arg Arg Gly Gln Pro Phe Ser Ile Thr Leu Gln Cys 35 40 45 Ser Asp Ser Leu Pro Pro Lys His His Leu Glu Leu Val Leu His 50 55 60 Leu Gly Lys Arg Asp Glu Val Val Ile Lys Val Gln Lys Glu His 65 70 75 Gly Ala Arg Asp Lys Trp Trp Phe Asn Gln Gln Gly Ala Gln Asp 80 85 90 Glu Ile Leu Leu Thr Leu His Ser Pro Ala Asn Ala Val Ile Gly 95 100 105 His Tyr Arg Leu Ala Val Leu Val Met Ser Pro Asp Gly His Ile 110 115 1 20 Val Glu Arg Ala Asp Lys Ile Ser Phe His Met Leu Phe Asn Pro 125 130 135 Trp Cys Arg Asp Asp Met Val Tyr Leu Pro Asp Glu Ser Lys Leu 140 145 150 Gln Glu Tyr Val Met Asn Glu Asp Gly Val Ile Tyr Met Gly Thr 155 160 165 Trp Asp Tyr Ile Arg Ser Ile Pro Trp Asn Tyr Gly Gln Phe Glu 170 175 180 Asp Tyr Val Met Asp Ile Cys Phe Glu Val Leu Asp Asn Ser Pro 185 190 195 Ala Ala Leu Lys Asn Ser Glu Met Asp Ile Glu His Arg Ser Asp 200 205 210 Pro Val Tyr Val Gly Arg Thr Ile Thr Ala Met Val Asn Ser Asn 215 220 225 Gly Asp Arg Gly Val Leu Thr Gly Arg Trp Glu Glu Pro Tyr Thr 230 235 240 Asp Gly Val Ala Pro Tyr Arg Trp Thr Gly Ser Val Pro Ile Leu 245 250 255 Gln Gln Trp Ser Lys Ala Gly Val Arg Pro Val Lys Tyr Gly Gln 260 265 270 Cys Trp Val Phe Ala Ala Val Ala Cys Thr Val Leu Arg Cys Leu 275 280 285 Gly Ile Pro Thr Arg Pro Ile Thr Asn Phe Ala Ser Ala His Asp 290 295 300 Val Asp Gly Asn Leu Ser Val Asp Phe Leu Leu Asn Glu Arg Leu 305 310 315 Glu Ser Leu Asp Ser Arg Gln Arg Ser Asp Ser Ser Trp Asn Phe 3 20 325 330 His Cys Trp Val Glu Ser Trp Met Ser Arg Glu Asp Leu Pro Glu 335 340 345 Gly Asn Asp Gly Trp Gln Val Leu Asp Pro Thr Pro Gln Glu Leu 350 355 360 Ser Asp Gly Glu Phe Cys Cys Gly Pro Cys Pro Val Ala Ala Ile 365 370 375 Lys Glu Gly Asn Leu Gly Val Lys Tyr Asp Ala Pro Phe Val Phe 380 385 390 Ala Glu Val Asn Ala Asp Thr Ile Tyr Trp Ile Val Gln Lys Asp 395 400 405 Gly Gln Arg Arg Lys Ile Thr Glu Asp His Ala Ser Val Gly Lys 410 415 420 Asn Ile Ser Thr Lys Ser Val Tyr Gly Asn His Arg Glu Asp Val 425 430 435 Thr Leu His Tyr Lys Tyr Pro Glu Gly Ser Gln Lys Glu Arg Glu 440 445 450 Val Tyr Lys Lys Ala Gly Arg Arg Val Thr Glu Pro Ser Asn Glu 455 460 465 Ile Ala Glu Gln Gly Arg Leu Gln Leu Ser Ile Lys His Ala Gln 470 475 480 Pro Val Phe Gly Thr Asp Phe Asp Val Ile Val Glu Val Lys Asn 485 490 495 Glu Gly Gly Arg Asp Ala His Ala Gln Leu Thr Met Leu Ala Met 500 505 510 Ala Val Thr Tyr Asn Ser Leu Arg Arg Gly Glu Cys Gln Arg Lys 515 520 525 Thr Ile Ser Val Thr Val Pro Ala His Lys Ala His Lys G lu Val 530 535 540 Met Arg Leu His Tyr Asp Asp Tyr Val Arg Cys Val Ser Glu His 545 550 555 His Leu Ile Arg Val Lys Ala Leu Leu Asp Ala Pro Gly Glu Asn 560 565 570 Gly Pro Ile Met Thr Val Ala Asn Ile Pro Leu Ser Thr Pro Glu 575 580 585 Leu Leu Val Gln Val Pro Gly Lys Ala Val Val Trp Glu Pro Leu 590 595 600 Thr Ala Tyr Val Ser Phe Thr Asn Pro Leu Pro Val Pro Leu Lys 605 610 615 Gly Gly Val Phe Thr Leu Glu Gly Ala Gly Leu Leu Ser Ala Thr 620 625 630 Gln Ile His Val Asn Gly Ala Val Ala Pro Ser Gly Lys Val Ser 635 640 645 Val Lys Leu Ser Phe Ser Pro Met Arg Thr Gly Val Arg Lys Leu 650 655 660 Leu Val Asp Phe Asp Ser Asp Arg Leu Lys Asp Val Lys Gly Val 665 670 675 Thr Thr Val Val Val His Lys Lys Tyr Arg Ser Leu Ile Thr Gly 680 685 690 Leu His Thr Asp SEQ ID NO: 3 Sequence Length: 695 Sequence Type: amino acid topology: linear sequence type: protein origin organism name: red sea bream liver sequence Met Ala Ser Tyr Lys Gly Leu Ile Val Asp Val Asn Gly Arg Ser 1 5 10 15 His Glu As n Asn Leu Ala His Arg Thr Arg Glu Ile Asp Arg Glu 20 25 30 Arg Leu Ile Val Arg Arg Gly Gln Pro Phe Ser Ile Thr Leu Gln 35 40 45 Cys Ser Asp Ser Leu Pro Pro Lys His His Leu Glu Leu Val Leu 50 55 60 His Leu Gly Lys Arg Asp Glu Val Val Ile Lys Val Gln Lys Glu 65 70 75 His Gly Ala Arg Asp Lys Trp Trp Phe Asn Gln Gln Gly Ala Gln 80 85 90 Asp Glu Ile Leu Leu Thr Leu His Ser Pro Ala Asn Ala Val Ile 95 100 105 Gly His Tyr Arg Leu Ala Val Leu Val Met Ser Pro Asp Gly His 110 115 120 Ile Val Glu Arg Ala Asp Lys Ile Ser Phe His Met Leu Phe Asn 125 130 135 Pro Trp Cys Arg Asp Asp Met Val Tyr Leu Pro Asp Glu Ser Lys 140 145 150 Leu Gln Glu Tyr Val Met Asn Glu Asp Gly Val Ile Tyr Met Gly 155 160 165 Thr Trp Asp Tyr Ile Arg Ser Ile Pro Trp Asn Tyr Gly Gln Phe 170 175 180 Glu Asp Tyr Val Met Asp Ile Cys Phe Glu Val Leu Asp Asn Ser 185 190 195 Pro Ala Ala Leu Lys Asn Ser Glu Met Asp Ile Glu His Arg Ser 200 205 210 Asp Pro Val Tyr Val Gly Arg Thr Ile Thr Ala Met Val Asn Ser 215 220 225 Asn Gly Asp Arg Gl y Val Leu Thr Gly Arg Trp Glu Glu Pro Tyr 230 235 240 Thr Asp Gly Val Ala Pro Tyr Arg Trp Thr Gly Ser Val Pro Ile 245 250 255 Leu Gln Gln Trp Ser Lys Ala Gly Val Arg Pro Val Lys Tyr Gly 260 265 270 Gln Cys Trp Val Phe Ala Ala Val Ala Cys Thr Val Leu Arg Cys 275 280 285 Leu Gly Ile Pro Thr Arg Pro Ile Thr Asn Phe Ala Ser Ala His 290 295 300 Asp Val Asp Gly Asn Leu Ser Val Asp Phe Leu Leu Asn Glu Arg 305 310 315 Leu Glu Ser Leu Asp Ser Arg Gln Arg Ser Asp Ser Ser Trp Asn 320 325 330 Phe His Cys Trp Val Glu Ser Trp Met Ser Arg Glu Asp Leu Pro 335 340 345 Glu Gly Asn Asp Gly Trp Gln Val Leu Asp Pro Thr Pro Gln Glu 350 355 360 Leu Ser Asp Gly Glu Phe Cys Cys Gly Pro Cys Pro Val Ala Ala 365 370 375 Ile Lys Glu Gly Asn Leu Gly Val Lys Tyr Asp Ala Pro Phe Val 380 385 390 Phe Ala Glu Val Asn Ala Asp Thr Ile Tyr Trp Ile Val Gln Lys 395 400 405 Asp Gly Gln Arg Arg Lys Ile Thr Glu Asp His Ala Ser Val Gly 410 415 420 Lys Asn Ile Ser Thr Lys Ser Val Tyr Gly Asn His Arg Glu Asp 425 430 435 Val Thr Le u His Tyr Lys Tyr Pro Glu Gly Ser Gln Lys Glu Arg 440 445 450 Glu Val Tyr Lys Lys Ala Gly Arg Arg Val Thr Glu Pro Ser Asn 455 460 465 Glu Ile Ala Glu Gln Gly Arg Leu Gln Leu Ser Ile Lys His Ala 470 475 480 Gln Pro Val Phe Gly Thr Asp Phe Asp Val Ile Val Glu Val Lys 485 490 495 Asn Glu Gly Gly Arg Asp Ala His Ala Gln Leu Thr Met Leu Ala 500 505 510 Met Ala Val Thr Tyr Asn Ser Leu Arg Arg Gly Glu Cys Gln Arg 515 520 525 Lys Thr Ile Ser Val Thr Val Pro Ala His Lys Ala His Lys Glu 530 535 540 Val Met Arg Leu His Tyr Asp Asp Tyr Val Arg Cys Val Ser Glu 545 550 555 His His Leu Ile Arg Val Lys Ala Leu Leu Asp Ala Pro Gly Glu 560 565 570 Asn Gly Pro Ile Met Thr Val Ala Asn Ile Pro Leu Ser Thr Pro 575 580 585 Glu Leu Leu Val Gln Val Pro Gly Lys Ala Val Val Trp Glu Pro 590 595 600 Leu Thr Ala Tyr Val Ser Phe Thr Asn Pro Leu Pro Val Pro Leu 605 610 615 Lys Gly Gly Val Phe Thr Leu Glu Gly Ala Gly Leu Leu Ser Ala 620 625 630 Thr Gln Ile His Val Asn Gly Ala Val Ala Pro Ser Gly Lys Val 635 640 645 Se r Val Lys Leu Ser Phe Ser Pro Met Arg Thr Gly Val Arg Lys 650 655 660 Leu Leu Val Asp Phe Asp Ser Asp Arg Leu Lys Asp Val Lys Gly 665 670 675 Val Thr Thr Val Val Val His Lys Lys Tyr Arg Ser Leu Ile Thr 680 685 690 Gly Leu His Thr Asp 695 SEQ ID NO: 4 Sequence length: 2085 Sequence type: Nucleic acid strand number: Double stranded topology: Linear sequence type: cDNA to mRNA Origin organism name: snapper liver Characteristic of the sequence Character that shows the characteristic: CDS Location: 1. . 2085 method to determine the characteristics: P sequence GCCAGCTACA AGGGGTTGAT TGTTGATGTG AATGGGAGAA GTCATGAAAA CAACTTGGCT 60 CACCGCACCA GGGAGATTGA TCGGGAGCGC CTGATCGTCC GCAGAGGTCA ACCCTTCTCC 120 ATCACTTTGC AGTGCTCTGA CTCTCTGCCG CCCAAACACC ACCTGGAGCT GGTCCTGCAC 180 CTCGGTAAGA GAGACGAGGT GGTGATCAAG GTTCAGAAGG AACATGGGGC CAGAGACAAG 240 TGGTGGTTTA ACCAGCAGGG AGCTCAGGAT GAAATACTGC TGACTCTGCA CAGCCCAGCG 300 AACGCTGTCA TTGGCCACTA CCGTCTGGCT GTGTTGGTGA TGTCACCAGA TGGTCACATC 360 GTAGAGAGGG CAGACAAAAT TAGCTTCCAC ATGCTCTTCA ACCCGTGGTG CAGAGATGAT 420 ATGGTTTACC TCCCTGATGA GAGTAAGCTC CAGGAGTATG TCATGAATGA AGATGGAGTG 480 ATTTACATGG GGACCTGGGA TTACATCAGA AGTATACCCT GGAATTATGG ACAGTTTGAG 540 GACTATGTGA TGGACATCTG TTTTGAAGTC TTGGACAACT CCCCAGCTGC CTTGAAAAAC 600 TCAGAGATGG ACATTGAGCA CAGATCAGAC CCCGTCTATG TCGGCAGGAC AATCACTGCA 660 ATGGTGAACT CTAACGGTGA CAGGGGTGTG TTGACTGGTC GCTGGGAGGA GCCGTACACT 720 GATGGGGTCG CACCGTATCG ATGGACCGGC AGCGTGCCGA TCCTCCAACA GTGGAGCAAG 780 GCCGGGGTGA GGCCGGTCAA ATATGGCCAG TGCTGGGTGT TTG CTGCCGT CGCCTGCACA 840 GTGCTGCGCT GCCTGGGAAT CCCAACACGC CCCATCACCA ACTTCGCTTC AGCCCATGAT 900 GTCGATGGTA ACCTCTCGGT AGACTTCCTG CTGAATGAGA GACTGGAGAG CTTGGACAGT 960 AGACAGAGAA GTGACAGTAG CTGGAACTTC CACTGTTGGG TTGAATCCTG GATGAGCAGA 1020 GAGGATCTCC CTGAAGGAAA TGATGGCTGG CAGGTTTTGG ATCCCACCCC TCAAGAACTG 1080 AGTGATGGTG AGTTTTGCTG TGGTCCGTGT CCAGTGGCGG CCATCAAGGA GGGAAATCTG 1140 GGAGTGAAGT ACGACGCCCC CTTTGTATTC GCTGAGGTGA ACGCTGACAC CATCTACTGG 1200 ATCGTCCAAA AAGATGGCCA ACGACGGAAG ATCACAGAGG ACCATGCTAG TGTGGGGAAG 1260 AACATCAGCA CAAAAAGCGT TTACGGCAAC CACAGAGAAG ATGTCACTCT GCACTACAAA 1320 TATCCTGAAG GCTCCCAGAA GGAGAGGGAA GTGTACAAGA AGGCGGGACG CCGGGTCACA 1380 GAGCCATCCA ACGAGATCGC AGAACAAGGA AGACTTCAGC TGTCAATCAA GCATGCCCAG 1440 CCTGTATTTG GGACAGACTT TGATGTGATT GTTGAGGTGA AGAATGAAGG AGGCAGAGAT 1500 GCTCATGCTC AGCTGACCAT GCTGGCCATG GCAGTAACTT ACAATTCTCT CCGCCGGGGG 1560 GAGTGCCAGA GAAAAACAAT CAGTGTGACT GTGCCCGCTC ACAAAGCCCA CAAGGAGGTT 1620 ATGCGTCTGC ACTACGACGA CTATGTCAGG TGTGTCTCTG AGCATCACCT G ATCAGGGTG 1680 AAAGCGCTCT TAGACGCTCC AGGGGAGAAC GGGCCCATCA TGACCGTGGC CAACATCCCA 1740 CTGAGCACGC CTGAACTCCT TGTACAGGTG CCTGGGAAGG CTGTTGTATG GGAACCACTG 1800 ACAGCCTACG TCTCCTTCAC CAATCCTCTG CCAGTTCCTC TGAAGGGTGG CGTTTTCACT 1860 TTGGAGGGTG CTGGCCTGCT GTCTGCCACT CAGATCCATG TTAATGGTGC TGTAGCTCCA 1920 AGTGGGAAAG TGTCTGTCAA GCTCTCTTTC TCCCCCATGC GCACCGGGGT GAGGAAGCTC 1980 CTGGTGGACT TTGACTCTGA CAGACTGAAG GACGTGAAGG GTGTCACCAC CGTGGTTGTC 2040 CACAAGAAAT ACAGATCTCT AATTACTGGA CTTCACACAG ACTAA 2085 SEQ ID NO: 5 Sequence length: 2520 Sequence type: Nucleic acid chain number: Double-stranded topology: Linear sequence type: cDNA to mRNA Origin organism name: Characteristics symbol of snapper sequence: CDS Location : 34. . 2121 Method of characterizing: P-sequence CTTTAACAGA CTTTGATAGG AAGAAGATCT GCG ATG GCC AGC TAC AAG GGG 51 Met Ala Ser Tyr Lys Gly 15 TTG ATT GTT GAT GTG AAT GGG AGA AGT CAT GAA AAC AAC TTG GCT 96 Leu Ile Val Asp Val Asn Gly Arg Ser His Glu Asn Asn Leu Ala 10 15 20 CAC CGC ACC AGG GAG ATT GAT CGG GAG CGC CTG ATC GTC CGC AGA 141 His Arg Thr Arg Glu Ile Asp Arg Glu Arg Leu Ile Val Arg Arg 25 30 35 GGT CAA CCC TTC TCC ATC ACT TTG CAG TGC TCT GAC TCT CTG CCG 186 Gly Gln Pro Phe Ser Ile Thr Leu Gln Cys Ser Asp Ser Leu Pro 40 45 50 CCC AAA CAC CAC CTG GAG CTG GTC CTG CAC CTC GGT AAG AGA GAC 231 Pro Lys His His Leu Glu Leu Val Leu His Leu Gly Lys Arg Asp 55 60 65 GAG GTG GTG ATC AAG GTT CAG AAG GAA CAT GGG GCC AGA GAC AAG 276 Glu Val Val Ile Lys Val Gln Lys Glu His Gly Ala Arg Asp Lys 70 75 80 TGG TGG TTT AAC CAG CAG GGA GCT CAG GAT GAA ATA CTG CTG ACT 321 Trp Trp Phe Asn Gln Gln Gly Ala Gln Asp Glu Ile Leu Leu Thr 85 90 95 CTG CAC AGC CCA GCG AAC GCT GTC ATT GGC CAC TAC CGT CTG GCT 366 Leu His Ser Pro Ala Asn Ala Val Ile Gly His Tyr Arg Leu Ala 100 105 110 GTG TTG GTG ATG TCA CCA GAT GGT CAC ATC GTA GAG AGG GCA GAC 411 Val Leu Val Met Ser Pro Asp Gly His Ile Val Glu Arg Ala Asp 115 120 125 AAA ATT AGC TTC CAC ATG CTC TTC AAC CCG TGG TGC AGA GAT GAT 456 Lys Ile Ser Phe His Met Leu Phe Asn Pro Trp Cys Arg Asp Asp 130 135 140 ATG GTT TAC CTC CCT GAT GAG AGT AAG CTC CAG GAG TAT GTC ATG 501 Met Val Tyr Leu Pro Asp Glu Ser Lys Leu Gln Glu Tyr Val Met 145 150 155 AAT GAA GAT GGA GTG ATT TAC TAC GTG GG ACC TGG GAT TAC ATC AGA 546 Asn Glu Asp Gly Val Ile Tyr Met Gly Thr Trp Asp Tyr Ile Arg 160 165 170 AGT ATA CCC TGG AAT TAT GGA CAG TTT GAG GAC TAT GTG ATG GAC 591 Ser Ile Pro Trp Asn Tyr Gly Gln Phe Glu Asp Tyr Val Met Asp 175 180 185 ATC TGT TTT GAA GTC TTG GAC AAC TCC CCA GCT GCC TTG AAA AAC 636 Ile Cys Phe Glu Val Leu Asp Asn Ser Pro Ala Ala Leu Lys Asn 190 195 200 TCA GAG ATG GAC ATT GAG CAC AGA TCA GAC CCC GTC TAT GTC GGC 681 Ser Glu Met Asp Ile Glu His Arg Ser Asp Pro Val Tyr Val Gly 205 210 215 AGG ACA ATC ACT GCA ATG GTG AAC TCT AAC GGT GAC AGG GGT GTG 726 Arg Thr Ile Thr Ala Met Val Asn Ser Asn Gly Asp Arg Gly Val 220 225 230 TTG ACT GGT CGC TGG GAG GAG CCG TAC ACT GAT GGG GTC GCA CCG 771 Leu Thr Gly Arg Trp Glu Glu Pro Tyr Thr Asp Gly Val Ala Pro 235 240 245 TAT CGA TGG ACC GGC AGC GTG CCG ATC CTC CAA CAG TGG AGC AAG 816 Tyr Arg Trp Thr Gly Ser Val Pro Ile Leu Gln Gln Trp Ser Lys 250 255 260 GCC GGG GTG AGG CCG GTC AAA TAT GGC CAG TGC TGG GTG TTT GCT 861 Ala Gly Val Arg Pro Val Lys Tyr Gly Gln Cys Trp Val Phe Ala 265 270 275 GCC GTC GCC TGC ACA GTG CTG CGC TGC CTG GGA ATC CCA ACA CGC 906 Ala Val Ala Cys Thr Val Leu Arg Cys Leu Gly Ile Pro Thr Arg 280 285 290 CCC ATC ACC AAC TTC GCT TCA GCC CAT GAT GTC GAT GGT AAC CTC 951 Pro Ile Thr Asn Phe Ala Ser Ala His Asp Val Asp Gly Asn Leu 295 300 305 TCG GTA GAC TTC CTG CTG AAT GAG AGA CTG GAG AGC TTG GAC AGT 996 Ser Val Asp Phe Leu Leu Asn Glu Arg Leu Glu Ser Leu Asp Ser 310 315 320 AGA CAG AGA AGT GAC AGT AGC TGG AAC TTC CAC TGT TGG GTT GAA 1041 Arg Gln Arg Ser Asp Ser Ser Trp Asn Phe His Cys Trp Val Glu 325 330 335 TCC TGG ATG AGC AGA GAG GAT CTC CCT GAA GGA AAT GAT GGC TGG 1086 Ser Trp Met Ser Arg Glu Asp Leu Pro Glu Gly Asn Asp Gly Trp 340 345 350 CAG GTT TTG GAT CCC ACC CCT CAA GAA CTG AGT GAT GGT GAG TTT 1131 Gln Val Leu Asp Pro Thr Pro Gln Glu Leu Ser Asp Gly Glu Phe 355 360 365 TGC TGT GGT CCG TGT CCA GTG GCG GCC ATC AAG GAG GGA AAT CTG 1176 Cys Cys Gly Pro Cys Pro Val Ala Ala Ile Lys Glu Gly Asn Leu 370 375 380 GGA GTG AAG TAC GAC GCC CCC TTT GTA TTC GCT GAG GTG AAC GCT 1221 Gly Val Lys Tyr Asp Ala Pro Phe Val Phe Ala Glu Val Asn Ala 385 390 395 GAC ACC ATC TAC TGG ATC GTC CAA AAA GAT GGC CAA CGA CGG AAG 1266 Asp Thr Ile Tyr Trp Ile Val Gln Lys Asp Gly Gln Arg Arg Lys 400 405 410 ATC ACA GAG GAC CAT GCT AGT GTG GGG AAG AAC ATC AGC ACA AAA 1311 Ile Thr Glu Asp His Ala Ser Val Gly Lys Asn Ile Ser Thr Lys 415 420 425 AGC GTT TAC GGC AAC CAC AGA GAA GAT GTC ACT CTG CAC TAC AAA 1356 Ser Val Tyr Gly Asn His Arg Glu Asp Val Thr Leu His Tyr Lys 430 435 440 TAT CCT GAA GGC TCC CAG AAG GAG AGG GAA GTG TAC AAG AAG GCG 1401 Tyr Pro Glu Gly Ser Gln Lys Glu Arg Glu Val Tyr Lys Lys Ala 445 450 455 GGA CGC CGG GTC ACA GAG CCA TCC AAC GAG ATC GCA GAA CAA GGA 1446 Gly Arg Arg Val Thr Glu Pro Ser Asn Glu Ile Ala Glu Gln Gly 460 465 470 AGA CTT CAG CTG TCA ATC AAG CAT GCC CAG CCT GTA TTT GGG ACA 1491 Arg Leu Gln Leu Ser Ile Lys His Ala Gln Pro Val Phe Gly Thr 475 480 485 GAC TTT GAT GTG ATT GTT GAG GTG AAG AAT GAA GGA GGC AGA GAT 1536 Asp Phe Asp Val Ile Val Glu Val Lys Asn Glu Gly Gly Arg Asp 490 495 500 GCT CAT GCT CAG CTG ACC ATG CTG GCC ATG GCA GTA ACT TAC AAT 1581 Ala His Ala Gln Leu Thr Met Leu Ala Met Ala Val Thr Tyr Asn 505 510 515 TCT CTC CGC CGG GGG GAG TGC CAG AGA AAA ACA ATC AGT GTG ACT 1626 Ser Leu Arg Arg Gly Glu Cys Gln Arg Lys Thr Ile Ser Val Thr 520 525 530 GTG CCC GCT CAC AAA GCC CAC AAG GAG GTT ATG CGT CTG CAC TAC 1671 Val Pro Ala His Lys Ala His Lys Glu Val Met Arg Leu His Tyr 535 540 545 GAC GAC TAT GTC AGG TGT GTC TCT GAG CAT CAC CTG ATC AGG GTG 1716 Asp Asp Tyr Val Arg Cys Val Ser Glu His His Leu Ile Arg Val 550 555 560 AAA GCG CTC TTA GAC GCT CCA GGG GAG AAC GGG CCC ATC ATG ACC 1761 Lys Ala Leu Leu Asp Ala Pro Gly Glu Asn Gly Pro Ile Met Thr 565 570 575 GTG GCC AAC ATC CCA CTG AGC ACG CCT GAA CTC CTT GTA CAG GTG 1806 Val Ala Asn Ile Pro Leu Ser Thr Pro Glu Leu Leu Val Gln Val 580 585 590 CCT GGG AAG GCT GTT GTA TGG GAA CCA CTG ACA GCC TAC GTC TCC 1851 Pro Gly Lys Ala Val Val Trp Glu Pro Leu Thr Ala Tyr Val Ser 595 600 605 TTC ACC AAT CCT CTG CCA GTT CCT CTG AAG GGT GGC GTT TTC ACT 1896 Phe Thr Asn Pro Leu Pro Val Pro Leu Lys Gly Gly Val Phe Thr 610 615 620 TTG GAG GGT GCT GGC CTG CTG TCT GCC ACT CAG ATC CAT GTT AAT 1941 Leu Glu Gly Ala Gly Leu Leu Ser Ala Thr Gln Ile His Val Asn 625 630 635 GGT GCT GTA GCT CCA AGT GGG AAA GTG TCT GTC AAG CTC TCT TTC 1986 Gly Ala Val Ala Pro Ser Gly Lys Val Ser Val Lys Leu Ser Phe 640 645 650 TCC CCC ATG C GC ACC GGG GTG AGG AAG CTC CTG GTG GAC TTT GAC 2031 Ser Pro Met Arg Thr Gly Val Arg Lys Leu Leu Val Asp Phe Asp 655 660 665 TCT GAC AGA CTG AAG GAC GTG AAG GGT GTC ACC ACC GTG GTT GTC 2076 Ser Asp Arg Leu Lys Asp Val Lys Gly Val Thr Thr Val Val Val 670 675 680 CAC AAG AAA TAC AGA TCT CTA ATT ACT GGA CTT CAC ACA GAC TAA 2121 His Lys Lys Tyr Arg Ser Leu Ile Thr Gly Leu His Thr Asp 685 690 695 AATAGACATA TCTTATATTA TGTGATTTTG TGACATTTCC TAGATGTGAG 2171 GTGGAGGTGA TGTATAAGGT AGATGATATC AACCGCTCAG TGTTATAACA 2221 GTTTATAATG CAAATAAGTT CCACTTAAAT GATACTGTAG CTATGTCCAC 2271 GAAGAAAATT CTTGACACAG TGTTAGTTTG ATTACCTTAA AGCCTTAAAG 2321 CCACTGTATG TCAGATGTGA ACTTGTCTGG CTTTGCATTA AAACCTGGCA 2371 CATGTTGCTC ACATGGAAAT GCACAGAAGC ACAACAGGTG ACGGCCTCTA 2421 GATGGAAAAT ATGTGCGTTT TGTTTCTGTT ACTCCTCTGT TTTATTGCCA 2471 AATTCAAGAT GCTTCCTTCT GTCTTCATTC CAAATGACTG CTGGTTTTT 2520 SEQ ID NO: 6 Sequence length: 695 Sequence type: Amino acid Topology: Linear Sequence type: Tamper Qualitative origin Biological name: Alas histara liver sequence Ala His Thr Asn Arg Leu Ile Ala Gly Val Asp Leu Arg Ser Gln 1 5 10 15 Glu Asn Asn Arg Glu His Arg Thr Glu Glu Ile Asp Arg Lys Arg 20 25 30 Leu Ile Val Arg Arg Arg Arg Gly Gln Ala Phe Ser Leu Thr Val His Leu 35 40 45 Ser Asp Pro Leu Gln Ser Gly His Glu Leu Ala Leu Val Leu Lys 50 55 60 Gln Asp Lys Asn Asn Asp Asp Ile Val Ile Arg Gln Arg Thr Ala 65 70 75 Gly Gly Ser Gly Asp Lys Trp Trp Leu His Gln Gln Ser Ala Arg 80 85 90 Asn Glu Leu Leu Leu Thr Val Tyr Ser Pro Ala Arg Ala Ala Val 95 100 105 Gly Glu Tyr Arg Leu Ala Val Glu Leu Met Ser Gly Asn Lys Leu 110 115 120 Leu Glu Arg Thr Asp Phe Thr Lys Met Tyr Leu Leu Phe Asn Pro 125 130 135 Trp Cys Lys Asp Asp Ala Val Tyr Leu Pro Asp Glu Ser Leu Leu 140 145 150 Lys Glu Tyr Ile Met Asn Glu Asn Gly Arg Ile Phe Thr Gly Ser 155 160 165 Ala Asp Trp Met Ser Gly Leu Pro Trp Asn Phe Gly Gln Phe Glu 170 175 180 Asp Asn Val Met Asp Ile Cys Phe Glu Ile Leu Asp Arg Phe Lys 185 190 195 Pro Ala Arg Ser Asp Pro Pr o Asn Asp Met Arg Gln Arg Trp Asp 200 205 210 Pro Val Tyr Ile Ser Arg Ala Val Val Ala Met Val Asn Ala Asn 215 220 225 Asp Asp Gly Gly Val Leu Val Gly Lys Trp Gln Glu Pro Tyr Thr 230 235 240 Gly Gly Val Gln Pro Thr Lys Trp Met Ser Ser Val Pro Ile Leu 245 250 255 Glu Lys Trp Ser Lys Ser Lys Ser Gly Val Lys Tyr Gly Gln Cys 260 265 270 Trp Val Phe Ala Ala Val Ala Cys Thr Val Leu Arg Cys Leu Gly 275 280 285 Ile Pro Thr Arg Cys Ile Thr Asn Phe Glu Ser Ala His Asp Thr 290 295 300 Asp Gly Asn Leu Ser Ile Asp Arg Val Tyr Asn Thr His Arg Gln 305 310 315 Ser Val Asn His Ala Asp Ser Ile Trp Asn Phe His Cys Trp Ile 320 325 330 Glu Ser Tyr Met Gln Arg Glu Asp Leu Pro Glu Gly Tyr Gly Gly 335 340 345 Trp Gln Val Leu Asp Pro Thr Pro Gln Glu Arg Ser Ser Gly Met 350 355 360 Phe Arg Cys Gly Pro Cys Pro Leu Lys Ala Ile Lys Glu Gly Asp 365 370 375 Leu Asn Val Lys Phe Asp Val Pro Phe Val Phe Ala Glu Val Asn 380 385 390 Ala Asp Ile Ile Asn Trp Glu Ile Arg Pro Asp Gly Gln Arg Met 395 400 405 Arg Leu Ser Ser As n Ser Ala Lys Val Gly Arg Asn Ile Ser Thr 410 415 420 Lys Ser Pro Tyr Ser Asn Glu Arg Glu Asp Ile Thr Leu Gln Tyr 425 430 435 Lys Tyr Gln Glu Gly Ser Ala Lys Glu Arg Glu Val Tyr Asn Lys 440 445 450 Ala Gly Arg Arg Ile Ser Gly Pro Asp Arg Glu Glu Glu Ser Lys 455 460 465 Pro Ala Asn Glu Pro Gly Asn Val Gln Leu Glu Ile Arg Tyr Ala 470 475 480 Lys Pro Val Phe Gly Thr Asp Phe Asp Val Ile Phe Glu Leu Glu 485 490 495 Asn Met Gly Asp Glu Glu Val Ser Cys Lys Leu Asn Met Met Ser 500 505 510 Lys Ala Val Thr Tyr Asn Ser Val His Leu Gly Glu Cys Gln Asn 515 520 525 Ser Thr Val Asn Val Val Ile Pro Ala His Lys Val His Arg Glu 530 535 540 Thr Val Arg Leu Leu Tyr Thr Lys Tyr Ala Ser Cys Val Ser Glu 545 550 555 His Asn Ile Ile Arg Val Val Gly Val Ala Arg Val Ser Gly Gln 560 565 570 Glu Lys Ser Ile Leu Glu Met Val Asn Ile Pro Leu Ser Lys Pro 575 580 585 Lys Leu Ser Ile Lys Val Pro Gly Trp Val Ile Leu Asn Arg Lys 590 595 600 Ile Thr Thr Val Ile Thr Phe Thr Asn Pro Leu Pro Val Pro Leu 605 610 615 Asn Arg Gl y Val Phe Thr Val Glu Gly Ala Gly Leu Leu Ser Thr 620 625 630 Lys Glu Ile Arg Ile Ser Gly Ser Ile Ala Pro Gly Gln Arg Val 635 640 645 Ser Val Glu Leu Ser Phe Thr Pro Met Arg Ala Gly Val Arg Glu 650 655 660 Phe Leu Val Asp Phe Asp Ser Asp Arg Leu Gln Asp Val Lys Gly 665 670 675 Val Ala Thr Leu Val Val Arg Lys Thr Ser Pro Ser Tyr Phe Pro 680 685 690 Met Pro Tyr Thr Leu 695 SEQ ID NO: 7 of sequence Length: 696 Sequence type: Amino acid topology: Linear sequence type: Protein origin Organism name: Alaska cod liver sequence Met Ala His Thr Asn Arg Leu Ile Ala Gly Val Asp Leu Arg Ser 1 5 10 15 Gln Glu Asn Asn Arg Glu His Arg Thr Glu Glu Ile Asp Arg Lys 20 25 30 Arg Leu Ile Val Arg Arg Gly Gln Ala Phe Ser Leu Thr Val His 35 40 45 Leu Ser Asp Pro Leu Gln Ser Gly His Glu Leu Ala Leu Val Leu 50 55 60 Lys Gln Asp Lys Asn Asn Asp Asp Ile Val Ile Arg Gln Arg Thr 65 70 75 Ala Gly Gly Ser Gly Asp Lys Trp Trp Leu His Gln Gln Ser Ala 80 85 90 Arg Asn Glu Leu Leu Leu Thr Val Tyr Ser Pro Ala Arg Ala Ala 95 100 105 Val Gly Glu Tyr Arg Leu Ala Val Glu Leu Met Ser Gly Asn Lys 110 115 120 Leu Leu Glu Arg Thr Asp Phe Thr Lys Met Tyr Leu Leu Phe Asn 125 130 135 Pro Trp Cys Lys Asp Asp Ala Val Tyr Leu Pro Asp Glu Ser Leu 140 145 150 Leu Lys Glu Tyr Ile Met Asn Glu Asn Gly Arg Ile Phe Thr Gly 155 160 165 Ser Ala Asp Trp Met Ser Gly Leu Pro Trp Asn Phe Gly Gln Phe 170 175 180 Glu Asp Asn Val Met Asp Ile Cys Phe Glu Ile Leu Asp Arg Phe 185 190 195 Lys Pro Ala Arg Ser Asp Pro Pro Asn Asp Met Arg Gln Arg Trp 200 205 210 Asp Pro Val Tyr Ile Ser Arg Ala Val Val Ala Met Val Asn Ala 215 220 225 Asn Asp Asp Gly Gly Val Leu Val Gly Lys Trp Gln Glu Pro Tyr 230 235 240 Thr Gly Gly Val Gln Pro Thr Lys Trp Met Ser Ser Val Pro Ile 245 250 255 Leu Glu Lys Trp Ser Lys Ser Lys Ser Gly Val Lys Tyr Gly Gln 260 265 270 Cys Trp Val Phe Ala Ala Val Ala Cys Thr Val Leu Arg Cys Leu 275 280 285 Gly Ile Pro Thr Arg Cys Ile Thr Asn Phe Glu Ser Ala His Asp 290 295 300 Thr Asp Gly Asn Leu S er Ile Asp Arg Val Tyr Asn Thr His Arg 305 310 315 Gln Ser Val Asn His Ala Asp Ser Ile Trp Asn Phe His Cys Trp 320 325 330 Ile Glu Ser Tyr Met Gln Arg Glu Asp Leu Pro Glu Gly Tyr Gly 335 340 345 Gly Trp Gln Val Leu Asp Pro Thr Pro Gln Glu Arg Ser Ser Gly 350 355 360 Met Phe Arg Cys Gly Pro Cys Pro Leu Lys Ala Ile Lys Glu Gly 365 370 375 Asp Leu Asn Val Lys Phe Asp Val Pro Phe Val Phe Ala Glu Val 380 385 390 Asn Ala Asp Ile Ile Asn Trp Glu Ile Arg Pro Asp Gly Gln Arg 395 400 405 Met Arg Leu Ser Ser Asn Ser Ala Lys Val Gly Arg Asn Ile Ser 410 415 420 Thr Lys Ser Pro Tyr Ser Asn Glu Arg Glu Asp Ile Thr Leu Gln 425 430 435 Tyr Lys Tyr Gln Glu Gly Ser Ala Lys Glu Arg Glu Val Tyr Asn 440 445 450 Lys Ala Gly Arg Arg Ile Ser Gly Pro Asp Arg Glu Glu Glu Ser 455 460 465 Lys Pro Ala Asn Glu Pro Gly Asn Val Gln Leu Glu Ile Arg Tyr 470 475 480 Ala Lys Pro Val Phe Gly Thr Asp Phe Asp Val Ile Phe Glu Leu 485 490 495 Glu Asn Met Gly Asp Glu Glu Val Ser Cys Lys Leu Asn Met Met 500 505 510 Ser Lys Ala V al Thr Tyr Asn Ser Val His Leu Gly Glu Cys Gln 515 520 525 Asn Ser Thr Val Asn Val Val Ile Pro Ala His Lys Val His Arg 530 535 540 Glu Thr Val Arg Leu Leu Tyr Thr Lys Tyr Ala Ser Cys Val Ser 545 550 555 Glu His Asn Ile Ile Arg Val Val Gly Val Ala Arg Val Ser Gly 560 565 570 Gln Glu Lys Ser Ile Leu Glu Met Val Asn Ile Pro Leu Ser Lys 575 580 585 Pro Lys Leu Ser Ile Lys Val Pro Gly Trp Val Ile Leu Asn Arg 590 595 600 Lys Ile Thr Thr Val Ile Thr Phe Thr Asn Pro Leu Pro Val Pro 605 610 615 Leu Asn Arg Gly Val Phe Thr Val Glu Gly Ala Gly Leu Leu Ser 620 625 630 Thr Lys Glu Ile Arg Ile Ser Gly Ser Ile Ala Pro Gly Gln Arg 635 640 645 Val Ser Val Glu Leu Ser Phe Thr Pro Met Arg Ala Gly Val Arg 650 655 660 Glu Phe Leu Val Asp Phe Asp Ser Asp Arg Leu Gln Asp Val Lys 665 670 675 Gly Val Ala Thr Leu Val Val Arg Lys Thr Ser Pro Ser Tyr Phe 680 685 690 Pro Met Pro Tyr Thr Leu 695 SEQ ID NO: 8 Sequence Length: 2088 Sequence Type: Nucleic Acid Number of Strands: Duplex Topology: Linear Sequence Species Class: cDNA to mRNA Origin organism name: Alaska pollack Character indicating liver sequence Character: Characteristic of CDS Location: 1. . 2088 method to determine the characteristics: P sequence GCCCACACAA ACCGTTTAAT TGCTGGTGTT GATCTGAGAA GCCAGGAAAA CAACCGGGAA 60 CACCGAACTG AGGAGATTGA TAGGAAGCGT TTGATTGTTC GGCGGGGACA AGCCTTCTCC 120 CTGACGGTGC ACCTCTCCGA CCCGCTGCAG TCCGGCCATG AGCTGGCCCT GGTCTTAAAG 180 CAGGATAAGA ACAACGATGA TATTGTGATC AGACAGCGAA CGGCTGGAGG GTCTGGTGAC 240 AAGTGGTGGT TACACCAGCA GAGCGCGAGG AACGAATTAC TGCTGACTGT GTACAGTCCT 300 GCCCGTGCTG CCGTTGGCGA GTACCGCTTG GCTGTTGAAC TGATGTCAGG GAATAAACTT 360 CTGGAGAGGA CGGACTTTAC CAAAATGTAC TTGCTGTTTA ATCCCTGGTG CAAAGATGAT 420 GCTGTGTACC TCCCTGATGA AAGTCTGCTC AAGGAATACA TTATGAACGA GAATGGTCGC 480 ATTTTCACTG GGAGTGCGGA TTGGATGAGT GGGTTGCCAT GGAATTTCGG ACAGTTTGAA 540 GACAATGTGA TGGACATCTG CTTTGAGATC CTTGACCGCT TTAAGCCAGC AAGGTCAGAC 600 CCCCCAAACG ACATGCGTCA GCGATGGGAC CCTGTCTACA TCAGCAGGGC AGTCGTTGCC 660 ATGGTGAATG CCAACGATGA CGGTGGAGTC TTGGTGGGGA AATGGCAGGA ACCTTACACA 720 GGTGGAGTAC AGCCAACCAA ATGGATGAGC AGTGTGCCCA TCCTGGAGAA GTGGAGCAAA 780 TCAAAGTCTG GAGTGAAGTA TGGCCAATGC TGGGTGTTTG C AGCCGTGGC CTGCACAGTG 840 CTGCGATGCC TGGGCATCCC CACACGCTGC ATCACCAACT TTGAGTCAGC CCATGACACA 900 GACGGAAACC TCTCCATCGA CCGAGTGTAC AACACACATA GGCAGAGTGT TAACCATGCT 960 GACAGCATCT GGAACTTTCA TTGTTGGATC GAGTCTTACA TGCAGAGAGA AGATCTACCT 1020 GAAGGATATG GTGGCTGGCA AGTCTTGGAC CCCACACCTC AGGAGAGGAG TAGTGGTATG 1080 TTTCGCTGTG GCCCATGTCC ATTGAAGGCC ATTAAAGAAG GGGACCTCAA TGTGAAGTTT 1140 GATGTTCCAT TTGTCTTTGC TGAGGTGAAT GCAGACATCA TCAATTGGGA AATCAGACCA 1200 GACGGTCAGC GAATGCGGCT TTCATCCAAC TCCGCAAAAG TGGGGAGGAA CATTAGCACC 1260 AAAAGTCCTT ACAGTAACGA GAGGGAAGAT ATAACCCTTC AGTACAAGTA CCAAGAAGGT 1320 TCAGCCAAGG AGCGGGAGGT GTACAACAAG GCAGGGCGGC GCATCTCCGG GCCGGATAGA 1380 GAAGAGGAAT CAAAACCAGC CAATGAACCA GGAAACGTGC AGCTGGAGAT CAGATACGCC 1440 AAGCCTGTGT TCGGGACCGA CTTTGACGTC ATCTTTGAGT TGGAGAACAT GGGAGACGAA 1500 GAAGTCAGCT GCAAATTGAA CATGATGTCA AAGGCTGTCA CGTATAACTC GGTCCACCTG 1560 GGAGAGTGCC AGAATAGCAC AGTCAATGTT GTCATTCCTG CTCACAAAGT CCACAGGGAG 1620 ACGGTGCGTC TACTCTACAC TAAGTATGCA TCGTGCGTCA GCGAACACAA CATCATCCGG 1680 GTGGTAGGGG TGGCAAGAGT GTCCGGCCAG GAAAAATCCA TCCTGGAGAT GGTCAACATC 1740 CCACTGAGCA AGCCCAAACT CAGTATTAAG GTTCCTGGCT GGGTGATTTT AAATAGGAAA 1800 ATCACCACCG TCATCACCTT CACCAATCCA TTGCCAGTGC CACTGAACCG AGGAGTGTTC 1860 ACTGTTGAAG GGGCTGGCCT ACTTTCAACC AAAGAGATCC GCATTTCTGG TAGCATCGCT 1920 CCAGGCCAGC GTGTGTCTGT GGAGCTGTCC TTCACACCCA TGAGGGCGGG GGTCAGGGAG 1980 TTCCTGGTGG ACTTTGACTC CGACAGGCTC CAGGACGTGA AGGGAGTCGC CACACTGGTG 2040 GTCCGCAAGA CTTCACCCTC CTATTTTCCC ATGCCCTACA CGTTGTGA 2088 SEQ ID NO: 9 Sequence length: 2921 Sequence type: Nucleic acid chain number: Double-stranded topology: Linear sequence type: cDNA to mRNA Origin organism name: Alaska pollack Liver sequence characteristic symbol: CDS existence Position: 32. . 2122 Method of characterizing: P-sequence AGCAACTCTT GGAAAGAATT TAGCAAAGAT A ATG GCC CAC ACA AAC 46 Met Ala His Thr Asn 1 5 CGT TTA ATT GCT GGT GTT GAT CTG AGA AGC CAG GAA AAC AAC CGG 91 Arg Leu Ile Ala Gly Val Asp Leu Arg Ser Gln Glu Asn Asn Arg 10 15 20 GAA CAC CGA ACT GAG GAG ATT GAT AGG AAG CGT TTG ATT GTT CGG 136 Glu His Arg Thr Glu Glu Ile Asp Arg Lys Arg Leu Ile Val Arg 25 30 35 CGG GGA CAA GCC TTC TCC CTG ACG GTG CAC CTC TCC GAC CCG CTG 181 Arg Gly Gln Ala Phe Ser Leu Thr Val His Leu Ser Asp Pro Leu 40 45 50 CAG TCC GGC CAT GAG CTG GCC CTG GTC TTA AAG CAG GAT AAG AAC 226 Gln Ser Gly His Glu Leu Ala Leu Val Leu Lys Gln Asp Lys Asn 55 60 65 AAC GAT GAT ATT GTG ATC AGA CAG CGA ACG GCT GGA GGG TCT GGT 271 Asn Asp Asp Ile Val Ile Arg Gln Arg Thr Ala Gly Gly Ser Gly 70 75 80 GAC AAG TGG TGG TTA CAC CAG CAG AGC GCG AGG AAC GAA TTA CTG 316 Asp Lys Trp Trp Leu His Gln Gln Ser Ala Arg Asn Glu Leu Leu 85 90 95 CTG ACT GTG TAC AGT CCT GCC CGT GCT GCC GTT GGC GAG TAC CGC 361 Leu Thr Val Tyr Ser Pro Ala Arg Ala Ala Val Gly Glu Tyr Arg 100 105 110 TTG GCT GTT GAA CTG ATG TCA GGG AAT AAA CTT CTG GAG AGG ACG 406 Leu Ala Val Glu Leu Met Ser Gly Asn Lys Leu Leu Glu Arg Thr 115 120 125 GAC TTT ACC AAA ATG TAC TTG CTG TTT AAT CCC TGG TGC AAA GAT 451 Asp Phe Thr Lys Met Tyr Leu Leu Phe Asn Pro Trp Cys Lys Asp 130 135 140 GAT GCT GTG TAC CTC CCT GAT GAA AGT CTG CTC AAG GAA TAC ATT 496 Asp Ala Val Tyr Leu Pro Asp Glu Ser Leu Leu Lys Glu Tyr Ile 145 150 155 ATG AAC GAG AAT GGT CGC ATT TTC ACT GGG AGT GCG GAT TGG ATG 541 Met Asn Glu Asn Gly Arg Ile Phe Thr Gly Ser Ala Asp Trp Met 160 165 170 AGT GGG TTG CCA TGG AAT TTC GGA CAG TTT GAA GAC AAT GTG ATG 586 Ser Gly Leu Pro Trp Asn Phe Gly Gln Phe Glu Asp Asn Val Met 175 180 185 GAC ATC TGC TTT GAG ATC CTT GAC CGC TTT AAG CCA GCA AGG TCA 631 Asp Ile Cys Phe Glu Ile Leu Asp Arg Phe Lys Pro Ala Arg Ser 190 195 200 GAC CCC CCA AAC GAC ATG CGT CAG CGA TGG GAC CCT GTC TAC ATC 676 Asp Pro Pro Asn Asp Met Arg Gln Arg Trp Asp Pro Val Tyr Ile 205 210 215 AGC AGG GCA GTC GTT GCC ATG GTG AAT GCC AAC GAT GAC GGT GGA 721 Ser Arg Ala Val Val Ala Met Val Asn Ala Asn Asp Asp Gly Gly 220 225 230 GTC TTG GTG GGG AAA TGG CAG GAA CCT TAC ACA GGT GGA GTA CAG 766 Val Leu Val Gly Lys Trp Gln Glu Pro Tyr Thr Gly Gly Val Gln 235 240 245 CCA ACC AAA TGG ATG AGC AGT GTG CCC ATC CTG GAG AAG TGG AGC 811 Pro Thr Lys Trp Met Ser Ser Val Pro Ile Leu Glu Lys Trp Ser 250 255 260 AAA TCA AAG TCT GGA GTG AAG TAT GGC CAA TGC TGG GTG TTT GCA 856 Lys Ser Lys Ser Gly Val Lys Tyr Gly Gln Cys Trp Val Phe Ala 265 270 275 GCC GTG GCC TGC ACA GTG CTG CGA TGC CTG GGC ATC CCC ACA CGC 901 Ala Val Ala Cys Thr Val Leu Arg Cys Leu Gly Ile Pro Thr Arg 280 285 290 TGC ATC ACC AAC TTT GAG TCA GCC CAT GAC ACA GAC GGA AAC CTC 946 Cys Ile Thr Asn Phe Glu Ser Ala His Asp Thr Asp Gly Asn Leu 295 300 305 TCC ATC GAC CGA GTG TAC AAC ACA CAT AGG CAG AGT GTT AAC CAT 991 Ser Ile Asp Arg Val Tyr Asn Thr His Arg Gln Ser Val Asn His 310 315 320 GCT GAC AGC ATC TGG AAC TTT CAT TGT TGG ATC GAG TCT TAC ATG 1036 Ala Asp Ser Ile Trp Asn Phe His Cys Trp Ile Glu Ser Tyr Met 325 330 335 CAG AGA GAA GAT CTA CCT GAA GGA TAT GGT GGC TGG CAA GTC TTG 1081 Gln Arg Glu Asp Leu Pro Glu Gly Tyr Gly Gly Trp Gln Val Leu 340 345 350 GAC CCC ACA CCT CAG GAG AGG AGT AGT GGT ATG TTT CGC TGT GGC 1126 Asp Pro Thr Pro Gln Glu Arg Ser Ser Gly Met Phe Arg Cys Gly 355 360 365 CCA TGT CCA TTG AAG GCC ATT AAA GAA GGG GAC CTC AAT GTG AAG 1171 Pro Cys Pro Leu Lys Ala Ile Lys Glu Gly Asp Leu Asn Val Lys 370 375 380 380 TTT GAT GTT CCA TTT GTC TTT GCT GAG GTG AAT GCA GAC ATC ATC 1216 Phe Asp Val Pro Phe Val Phe Ala Glu Val Asn Ala Asp Ile Ile 385 390 395 AAT TGG GAA ATC AGA CCA GAC GGT CAG CGA ATG CGG CTT TCA TCC 1261 Asn Trp Glu Ile Arg Pro Asp Gly Gln Arg Met Arg Leu Ser Ser 400 405 410 AAC TCC GCA AAA GTG GGG AGG AAC ATT AGC ACC AAA AGT CCT TAC 1306 Asn Ser Ala Lys Val Gly Arg Asn Ile Ser Thr Lys Ser Pro Tyr 415 420 425 AGT AAC GAG AGG GAA GAT ATA ACC CTT CAG TAC AAG TAC CAA GAA 1351 Ser Asn Glu Arg Glu Asp Ile Thr Leu Gln Tyr Lys Tyr Gln Glu 430 435 440 GGT TCA GCC AAG GAG CGG GAG GTG TAC AAC AAG GCA GGG CGG CGC 1396 Gly Ser Ala Lys Glu Arg Glu Val Tyr Asn Lys Ala Gly Arg Arg 445 450 455 ATC TCC GGG CCG GAT AGA GAA GAG GAA TCA AAA CCA GCC AAT GAA 1441 Ile Ser Gly Pro Asp Arg Glu Glu Glu Ser Lys Pro Ala Asn Glu 460 465 470 CCA GGA AAC GTG CAG CTG GAG ATC AGA TAC GCC AAG CCT GTG TTC 1486 Pro Gly Asn Val Gln Leu Glu Ile Arg Tyr Ala Lys Pro Val Phe 475 480 485 GGG ACC GAC TTT GAC GTC ATC TTT GAG TTG GAG AAC ATG GGA GAC 1531 Gly Thr Asp Phe Asp Val Ile Phe Glu Leu Glu Asn Met Gly Asp 490 495 500 GAA GAA GTC AGC TGC AAA TTG AAC ATG ATG TCA AAG GCT GTC ACG 1576 Glu Glu Val Ser Cys Lys Leu Asn Met Met Ser Lys Ala Val Thr 505 510 515 TAT AAC TCG GTC CAC CTG GGA GAG TGC CAG AAT AGC ACA GTC AAT 1621 Tyr Asn Ser Val His Leu Gly Glu Cys Gln Asn Ser Thr Val Asn 520 525 530 GTT GTC ATT CCT GCT CAC AAA GTC CAC AGG GAG ACG GTG CGT CTA 1666 Val Val Ile Pro Ala His Lys Val His Arg Glu Thr Val Arg Leu 535 540 545 CTC TAC ACT AAG TAT GCA TCG TGC GTC AGC GAA CAC AAC ATC ATC 1711 Leu Tyr Thr Lys Tyr Ala Ser Cys Val Ser Glu His Asn Ile Ile 550 555 560 CGG GTG GTA GGG GTG GCA AGA GTG TCC GGC CAG GAA AAA TCC ATC 1756 Arg Val Val Gly Val Ala Arg Val Ser Gly Gln Glu Lys Ser Ile 565 570 575 CTG GAG ATG GTC AAC ATC CCA CTG AGC AAG CCC AAA CTC AGT ATT 1801 Leu Glu Met Val Asn Ile Pro Leu Ser Lys Pro Lys Leu Ser Ile 580 585 590 AAG GTT CCT GGC TGG GTG ATT TTA AAT AGG AAA ATC ACC ACC GTC 1846 Lys Val Pro Gly Trp Val Ile Leu Asn Arg Lys Ile Thr Thr Val 595 600 605 ATC ACC TTC ACC AAT CCA TTG CCA GTG CCA CTG AAC CGA GGA GTG 1891 Ile Thr Phe Thr Asn Pro Leu Pro Val Pro Leu Asn Arg Gly Val 610 615 620 TTC ACT GTT GAA GGG GCT GGC CTA CTT TCA ACC AAA GAG ATC CGC 1936 Phe Thr Val Glu Gly Ala Gly Leu Leu Ser Thr Lys Glu Ile Arg 625 630 635 ATT TCT GGT AGC ATC GCT CCA GGC CAG CGT GTG TCT GTG GAG CTG 1981 Ile Ser Gly Ser Ile Ala Pro Gly Gln Arg Val Ser Val Glu Leu 640 645 650 TCC TTC ACA CCC ATG A GG GCG GGG GTC AGG GAG TTC CTG GTG GAC 2026 Ser Phe Thr Pro Met Arg Ala Gly Val Arg Glu Phe Leu Val Asp 655 660 665 TTT GAC TCC GAC AGG CTC CAG GAC GTG AAG GGA GTC GCC ACA CTG 2071 Phe Asp Ser Asp Arg Leu Gln Asp Val Lys Gly Val Ala Thr Leu 670 675 680 GTG GTC CGC AAG ACT TCA CCC TCC TAT TTT CCC ATG CCC TAC ACG 2116 Val Val Arg Lys Thr Ser Pro Ser Tyr Phe Pro Met Pro Tyr Thr 685 690 695 TTG TGATCAAACC TATAGCTGTC AACAGGGCTC TGGCACTCAT TCTTATACTA 2169 Leu 696 ACAAATATAT TTAGCAAAGT CAAGCAAGGG TTTCACTTTT CTTAATATAC 2219 CATGATGTGT AGCGCTGATT CAATTAATGA ATAAATTAAT TTCAATTAAT 2269 GTGAAGAAAA TGCAAACATT GCCTTAATTC TTTGCAATGT CACAGGAATA 2319 GCGTAAATCA TGGCTCATTG ATATTAAATG TAGTATTGAC ATATATCCAT 2369 GCATTTTGCA CTTCTGCAAA TCACCATTTT GTTGTTAATC AATGTTTTAC 2419 CACGATTTTT GCATCTATTC TTGTTTAATT GTAATCAAGA CATTTACATG 2469 ATTGTGGGGG CCAAAGTATA TAGATGTTGT GGTTGGGAAA TGGGGCAATA 2519 ATAGGGGAAG GGTTAATTAT AGGGTCAGTG TTAGTAATTG GTTAAGGTTA 2569 CTAATAGGGT AAGTGTTACA GTGTAAAGAT AAGCCTTTGA TTTTGTTAAA 261 9 TTTATTATGC CTTTCATCAA CAGTGGTTTG GGGTTTTATA ACAACAATTA 2669 AAGTGCTTAA CTACTGGTGA ACGACGTTGC AGAACGTATA TGGTACAAGT 2719 TTGTGTTGAT CGCATGGAAA AGGGAATAAC CAGTTACAAC TTATATGGTA 2769 AGAGCCTGGT AATACCATGG AAACAAACGA GGCTTCCTTT TACAGTACAG 2819 TTTCAGCGTC ATGAATATTT GGCCTGTTAA GCCCTTTGAG ACTGTAATGG 2869 TGATTAAGGG CTATACAAAT AAAATTGAAT TGAATTGAAT TAAAAAAAAA 2919 AA 2921 SEQ ID NO: 10 SEQ Length: 29 sequence Type: number of nucleic acid strands: single-stranded topology: type of linear sequence: other nucleic acid synthetic DNA sequence GTCAAGTACG GCCAGTGCTG GGTCTTCGC
29 SEQ ID NO: 11 Sequence length: 8 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence Tyr Gly Gln Cys Trp Val Phe Ala 8 SEQ ID NO: 12 Sequence length: 30 Sequence type: Number of nucleic acid strands: Single-stranded topology: Type of linear sequence: Other nucleic acid Synthetic DNA Characteristic character of sequence: RBS Location: 15..23 Method of characterizing feature: S Intended function: Specification In the book, the chemically synthesized DNA-1 in Figure 4 is constructed.
Sequence of idealized RBS AATTCATCGA TTAGTAAGGA GGTTTAAAAT 30 SEQ ID NO: 13 Sequence length: 30 Sequence type: Nucleic acid strand number: Single-stranded topology: Linear sequence type: Other nucleic acid Synthetic DNA Sequence characteristics Characteristic symbol: CDS Location: 1..30 Method for determining characteristic: P Intended function: In the specification, chemically synthesized DNA-1 in FIG. 4 is constructed.
N-terminal region of adult sea bream transglutaminase encodes the downstream nucleotide sequence of SEQ ID NO: 12 Sequence GGCTTCTTAT AAAGGTCTGA TTGTTGATGT 30 SEQ ID NO: 14 Sequence length: 32 Sequence type: Nucleic acid chain number: Single-stranded topology: Linear Type of sequence: other nucleic acid Synthetic DNA Symbol representing the characteristic of sequence: CDS Location: 1..32 Method of determining the characteristic: P Intended function: in the specification, chemically synthesized DNA-1 in FIG. Construct
N-terminal region of adult sea bream transglutaminase encodes the downstream nucleotide sequence of SEQ ID NO: 13 Sequence TAATGGTCGT TCTCATGAAA ACAACCTGGC AC 32 SEQ ID NO: 15 Sequence length: 33 Sequence type: Nucleic acid chain number: Single-stranded topology: direct Type of chained sequence: Other nucleic acid Synthetic DNA Symbol representing the characteristic of the sequence: CDS Location: 1..33 Method for determining the characteristic: P Intended function: In the specification, chemically synthesized DNA- Construct 1
N-terminal region of adult sea bream transglutaminase encodes the downstream nucleotide sequence of SEQ ID NO: 14 Sequence ATCGTACGCG TGAAATCGAC CGTGAGCGCC TGA 33 SEQ ID NO: 16 Sequence length: 30 Sequence type: Nucleic acid chain number: Single-stranded topology: direct Type of chain sequence: Other nucleic acid Synthetic DNA Intended function: In the specification, chemically synthesized DNA-1 in FIG.
Complementary strand role of SEQ ID NO: 12 Sequence AGCCATTTTA AACCTCCTTA CTAATCGATG 30 SEQ ID NO: 17 Sequence length: 30 Sequence type: Nucleic acid strand number: Single-stranded topology: Linear sequence type: Other nucleic acid Synthetic DNA Intended function: In the specification, the chemically synthesized DNA-1 in Fig. 4 is constructed.
Complementary strand role of SEQ ID NO: 13 Sequence ATTAACATCA ACAATCAGAC CTTTATAAGA 30 SEQ ID NO: 18 Sequence length: 32 Sequence type: Nucleic acid strand number: Single-stranded topology: Linear sequence type: Other nucleic acid Synthetic DNA Intended function: In the specification, the chemically synthesized DNA-1 in Fig. 4 is constructed.
Complementary strand role of SEQ ID NO: 14 Sequence CGATGTGCCA GGTTGTTTTC ATGAGAACGA CC 32 SEQ ID NO: 19 Sequence length: 33 Sequence type: Nucleic acid strand number: Single-stranded topology: Linear sequence type: Other nucleic acid synthesis DNA Intended function: In the specification, the chemically synthesized DNA-1 in FIG.
Complementary strand role of SEQ ID NO: 15 Sequence AGCTTCAGGC GCTCACGGTC GATTTCACGC GTA 33

SEQ ID NO: 20 Sequence Length: 7 Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Fragment Type: Intermediate Fragment Origin Organism Name: Teragra chalcogramma Tissue Type: Liver SEQ ID NO: 21 sequence length: 7 sequence type: amino acid topology: linear sequence type: peptide fragment type: intermediate fragment origin organism name: Teragra chalcogramma tissue type: liver SEQ ID NO: 22 sequence length: 7 sequence type: amino acid topology: linear sequence type: peptide fragment type: intermediate fragment origin organism name: Teragra chalcogramma tissue type: liver SEQ ID NO: 23 Sequence length: 8 Sequence type: Amino acid Topology: Linear Sequence type: Peptide fragment type: Intermediate fragment Origin organism name: Teragra chalcogramma Tissue type: Liver SEQ ID NO: 24 Sequence Length: 13 Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Fragment Type: Intermediate Fragment Origin Organism Name: Teragra chalcogramma Tissue Type: Liver Sequence Phe Asp Val Pro Phe Val Phe Ala Glu Val Asn Ala Asp 1 5 10 SEQ ID NO: 25 Sequence Length: 6 Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Fragment Type: Intermediate Fragment Origin Organism Name: Teragra chalcogramma Tissue Type: liver SEQ ID NO: 26 sequence length: 10 sequence type: amino acid topology: linear sequence type: peptide fragment type: intermediate fragment origin organism name: Pagrus major tissue type: liver SEQ ID NO: 27 Sequence Length: Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Fragment Type: Intermediate Fragment Origin Organism Name: Pagrus major Tissue Type: Liver Sequence Xaa Xaa Phe Asn Gln Gln Gly Ala Gln Asp Glu Ile Leu Leu Leu Thr 1 5 10 15 Leu His SEQ ID NO: 28 Sequence Length: 9 Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Fragment Type: Intermediate Fragment Origin Organism Name: Pagrus major Tissue type: liver SEQ ID NO: 29 sequence length: 16 sequence type: amino acid topology: linear sequence type: peptide fragment type: intermediate fragment origin organism name: Pagrus major tissue type: liver sequence Leu Gln Glu Tyr Val Met Asn Glu Asp Gly Val Ile Tyr Met Gly 1 5 10 15 Thr SEQ ID NO: 30 Sequence Length: 18 Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Fragment Type: Intermediate Fragment Origin Organism Name: Pagrus major Tissue type: Liver sequence Asn Ser Glu Met Asp Ile Glu His Arg Ser Asp Pro Val Tyr Val 1 5 10 15 Gly Arg Thr SEQ ID NO: 31 Sequence length: 16 Sequence type: Amino acid topology: Linear Sequence type: Peptide fragment type: Intermediate fragment Origin organism name: Pagrus major Tissue type: Liver sequence Tyr Asp Ala Pro Phe Val Phe Ala Glu Val Asn Ala Asp Thr Ile 1 5 10 15 Tyr SEQ ID NO: 32 of sequence Length: 14 array : Amino acid topology: Linear sequence type: Peptide fragment type: Intermediate fragment Origin organism name: Pagrus major Tissue type: Liver sequence Ser Val Tyr Gly Asn His Arg Glu Asp Val Thr Leu His Tyr 1 5 10 SEQ ID NO: : 33 Sequence Length: 18 Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Fragment Type: Intermediate Fragment Origin Organism Name: Pagrus major Tissue Type: Liver Sequence Ala Gly Arg Arg Val Thr Glu Pro Ser Asn Glu Ile Ala Glu Gln 1 5 10 15 Gly Arg Leu SEQ ID NO: 34 Sequence Length: 15 Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Fragment Type: Intermediate Fragment Origin Organism Name: Pagrus major Tissue type: Liver sequence Xaa Ala Gln Pro Val Phe Gly Thr Asp Phe Asp Val Ile Val Glu 1 5 10 15 SEQ ID NO: 35 Sequence length: 17 Sequence type: Amino acid Topology: Linear sequence type : Bae Tide fragment type: Intermediate fragment Origin organism name: Pagrus major Tissue type: Liver sequence Asn Glu Gly Gly Arg Asp Ala His Ala Gln Leu Thr Xaa Leu Ala 1 5 10 15 Xaa Ala SEQ ID NO: 36 Sequence length: 9 Sequence type: Amino acid topology: Linear sequence type: Peptide fragment type: Intermediate fragment Origin organism name: Pagrus major Tissue type: Liver SEQ ID NO: 37 sequence length: 9 sequence type: amino acid topology: linear sequence type: peptide fragment type: intermediate fragment origin organism name: Pagrus major tissue type: liver SEQ ID NO: 38 Sequence Length: 18 Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Fragment Type: Intermediate Fragment Origin Organism Name: Pagrus major Tissue Type: Liver Sequence Gly Gly Val Phe Thr Leu Glu Gly Ala Gly Leu Leu Ser Ala Thr 1 5 10 15 Gln Ile His SEQ ID NO: 39 Sequence Length: 11 Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Fragment Type: Intermediate Fragment Origin Organism Name: Pagrus major Tissue type: Liver SEQ ID NO: 40 sequence length: 10 sequence type: amino acid topology: linear sequence type: peptide fragment type: intermediate fragment origin organism name: Pagrus major tissue type: liver SEQ ID NO: 41 Sequence Length: 8 Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Fragment Type: Intermediate Fragment Origin Organism Name: Pagrus major Tissue Type: Liver SEQ ID NO: 42 sequence length: 11 sequence type: amino acid topology: linear sequence type: peptide fragment type: intermediate fragment origin organism name: Pagrus major tissue type: liver SEQ ID NO: 43 sequence length: 687 sequence type: amino acid topology: linear sequence type: protein origin organism name: flounder liver sequence Asp Asn Gln Asn Ile Pro Ile Thr Asp Val Asp Val Arg Ser His 1 5 10 15 Glu Asn Asn Leu Ala His Arg Thr Arg Glu Ile Asp Arg Glu Arg 20 25 30 Leu Ile Val Arg Arg Gly Gln Pro Phe Ser Ile Ser Leu Gln Cys 35 40 45 Cys Asp Ser Leu Thr Arg Asn His His Leu Glu Leu Ser Leu His 50 55 60 Leu Gly Lys Lys Asp Glu Val Val Ile Lys Val His Asn Glu Pro 65 70 75 Glu Ala Gly Gly Lys Trp Trp Phe Asn His Gln Lys Val Gln Asp 80 85 90 Glu Ile Leu Leu Thr Leu His Ser Pro Ala Asp Ala Ile Ile Gly 95 100 105 Glu Tyr His Leu Thr Val Leu Ile Lys Ser Pro Asp Gly His Phe 110 115 120 Val Lys Lys Thr Lys Asn Ile Gly Phe His Leu Leu Phe Asn Pro 125 130 135 Trp Cys Lys Asp Asp Ala Val Tyr Leu Pro Asp Glu Arg Met Leu 140 145 150 Asp Glu Tyr Val Met Asn Glu Glu Gly Ile Ile Tyr Arg Gly Thr 155 160 165 Ser Asn His Ile Ser Ser Ile Pro Trp Asn Tyr Gly Gln Phe Glu 170 175 180 Asp Tyr Val Met Asp Ile Cys Phe Gln Val Leu Asp Asn Ser Lys 185 190 195 Glu Ala Leu Lys Asn Ser Lys Met Asp Ile Glu Lys Arg Ser Asp 200 205 210 Pro Val Tyr Val Ser Arg Met Ile Thr Ala Met Val Asn Ser Asn 215 220 225 Gly Asp Arg Gly Val Leu Thr Gly Gln Trp His Glu Pro Tyr Thr 230 235 240 Gly Gly Phe Ser Pro Leu Arg Trp Thr Gly Ser Val Pro Ile Leu 245 250 255 Arg Lys Trp Ser Lys Ala Glu Val Arg Ala Val Lys Tyr Gly Gln 260 265 270 Cys Trp Val Phe Ala Ala Val Ala Cys Thr Val Leu Arg Cys Leu 275 280 285 Gly Ile Pro Thr Arg Asn Ile Thr Asn Phe Asn Ser Ala His Asp 290 295 300 Val Asp Gly Asn Leu Ser Val Asp Ile Val Leu Asn Lys Glu Met 305 310 315 Glu Ser Val Gly Lys Lys Asp Ser Ser Trp Asn Phe His Cys Trp 320 325 330 Ile Glu Ser Trp Met Arg Arg Asp Asp Leu Ser Lys Gly Asn Asp 335 340 345 Gly Trp Gln Val Leu Asp Pro Thr Pro Gln Glu Leu Ser Asp Gly 350 355 360 Glu Tyr Cys Cys Gly Pro Cys Pro Val Thr Ala Ile Lys Glu Gly 365 370 375 Asn Leu Ser Val Lys Tyr Asp Ala Pro Phe Ile Phe Ala Glu Val 380 385 390 Asn Ala Asp Ile Ile Tyr Trp Met Ala Gly Pro Gly Gly Glu Arg 395 400 405 Lys Lys Ile Asp Val Asp Gln Ser Gly Val Gly Lys Asn Ile Ser 410 415 420 Thr Lys Ser Leu Tyr Gly Asp Tyr Arg Glu Asp Val Thr Leu His 425 430 435 Tyr Lys Tyr Pro Glu Gly Ser Lys Lys Glu Arg Glu Val Tyr Gln 440 445 450 Lys Ala Gly His Arg Ile Lys Glu Gln Ile Cys Glu Asn Lys Gly 455 460 465 Pro Gln Gln Leu Gln Leu Ser Val Lys His Gly Lys Pro Val Phe 470 475 480 Gly Thr Asp Phe Asp Val Ile Val Glu Val Lys Asn Glu Gly Gln 485 490 495 Lys Asp Thr Ser Pro Gln Leu Leu Ile Val Val Met Ala Val Thr 505 510 510 Tyr Asn Ser Ile Asn Gln Gly Glu Cys Gln Arg Lys Ala Thr Ile 515 520 525 Val Thr Val Pro Ala Arg Lys Thr His Lys Glu Val Leu Arg Leu 530 535 540 Arg Tyr Asp Asp Tyr Val Lys Cys Val Ser Glu His His Leu Ile 545 550 555 Arg Val Lys Ala Leu Met Glu Val Pro Gly Asp Asn Lys Pro Val 560 565 570 Met Ser Val Ala Asn Ile Pro Leu Ser Met Pro Glu Leu Leu Val 575 580 585 Glu Val Pro Gly Ser Ile Ile Val Gln Glu Lys Val Thr Ala Phe 590 595 600 Ile Ser Phe Thr Asn Pro Leu Thr Val Pro Leu Lys Arg Gly Met 605 610 615 Phe Thr Val Glu Gly Ser Gly Leu Leu Ser Ala Ser Glu Ile Tyr 620 625 630 Val Lys Gly Asp Ile Ala Pro Gly Gln Lys Val Ser Val Lys Ile 635 640 645 Thr Phe Thr Pro Met Arg Val Gly Val Arg Lys Leu Leu Val Asp 650 655 660 Phe Asp Ser Asp Arg Leu Lys Asp Val Lys Gly Val Thr Thr Val 665 670 675 Val Val Arg Lys Lys Ser Cys Phe Ile Arg Cys Pro 680 685 SEQ ID NO: 44 Sequence Length: 688 Sequence Type: Amino Acid Topology: Linear Sequence Type: Protein Origin Biological Name: Flounder Liver Sequence Met Asp Asn Gln Asn Ile Pro Ile Thr Asp Val Asp Val Arg Ser 1 5 10 15 His Glu Asn Asn Leu Ala His Arg Thr Arg Glu Ile Asp Arg Glu 20 25 30 Arg Leu Ile Val Arg Arg Gly Gln Pro Phe Ser Ile Ser Leu Gln 35 40 45 Cys Cys Asp Ser Leu Thr Arg Asn His His Leu Glu Leu Ser Leu 50 55 60 His Leu Gly Lys Lys Asp Glu Val Val Ile Lys Val His Asn Glu 65 70 75 Pro Glu Ala Gly Gly Lys Trp Trp Phe Asn His Gln Lys Val Gln 80 85 90 Asp Glu Ile Leu Leu Thr Leu His Ser Pro Ala Asp Ala Ile Ile 95 100 105 Gly Glu Tyr His Leu Thr Val Leu Ile Lys Ser Pro Asp Gly His 110 115 120 Phe Val Lys Lys Thr Lys Asn Ile Gly Phe His Leu Leu Phe Asn 125 130 135 Pro Trp Cys Lys Asp Asp Ala Val Tyr Leu Pro Asp Glu Arg Met 140 145 150 Leu Asp Glu Tyr Val Met Asn Glu Glu Gly Ile Ile Tyr Arg Gly 155 160 165 Thr Ser Asn His Ile Ser Ser Ile Pro Trp Asn Tyr Gly Gln Phe 170 175 180 Glu Asp Tyr Val Met Asp Ile Cys Phe Gln Val Leu Asp Asn Ser 185 190 195 Lys Glu Ala Leu Lys Asn Ser Lys Met Asp Ile Glu Lys Arg Ser 200 205 210 Asp Pro Val Tyr Val Ser Arg Met Ile Thr Ala Met Val Asn Ser 215 220 225 Asn Gly Asp Arg Gly Val Leu Thr Gly Gln Trp His Glu Pro Tyr 230 235 240 Thr Gly Gly Phe Ser Pro Leu Arg Trp Thr Gly Ser Val Pro Ile 245 250 255 Leu Arg Lys Trp Ser Lys Ala Glu Val Arg Ala Val Lys Tyr Gly 260 265 270 Gln Cys Trp Val Phe Ala Ala Val Ala Cys Thr Val Leu Arg Cys 275 280 285 Leu Gly Ile Pro Thr Arg Asn Ile Thr Asn Phe Asn Ser Ala His 290 295 300 Asp Val Asp Gly Asn Leu Ser Val Asp Ile Val Leu Asn Lys Glu 305 310 315 Met Glu Ser Val Gly Lys Lys Asp Ser Ser Trp Asn Phe His Cys 320 325 330 Trp Ile Glu Ser Trp Met Arg Arg Asp Asp Leu Ser Lys Gly Asn 335 340 345 Asp Gly Trp Gln Val Leu Asp Pro Thr Pro Gln Glu Leu Ser Asp 350 355 360 Gly Glu Tyr Cys Cys Gly Pro Cys Pro Val Thr Ala Ile Lys Glu 365 370 375 Gly Asn Leu Ser Val Lys Tyr Asp Ala Pro Phe Ile Phe Ala Glu 380 385 390 Val Asn Ala Asp Ile Ile Tyr Trp Met Ala Gly Pro Gly Gly Glu 395 400 405 Arg Lys Lys Ile Asp Val Asp Gln Ser Gly Val Gly Lys Asn Ile 410 415 420 Ser Thr Lys Ser Leu Tyr Gly Asp Tyr Arg Glu Asp Val Thr Leu 425 430 435 His Tyr Lys Tyr Pro Glu Gly Ser Lys Lys Glu Arg Glu Val Tyr 440 445 450 Gln Lys Ala Gly His Arg Ile Lys Glu Gln Ile Cys Glu Asn Lys 455 460 465 Gly Pro Gln Gln Leu Gln Leu Ser Val Lys His Gly Lys Pro Val 470 475 480 Phe Gly Thr Asp Phe Asp Val Ile Val Glu Val Lys Asn Glu Gly 485 490 495 Gln Lys Asp Thr Ser Pro Gln Leu Leu Ile Val Val Met Ala Val 500 505 510 Thr Tyr Asn Ser Ile Asn Gln Gly Glu Cys Gln Arg Lys Ala Thr 515 520 525 Ile Val Thr Val Pro Ala Arg Lys Thr His Lys Glu Val Leu Arg 530 535 540 Leu Arg Tyr Asp Asp Tyr Val Lys Cys Val Ser Glu His His Leu 545 550 555 Ile Arg Val Lys Ala Leu Met Glu Val Pro Gly Asp Asn Lys Pro 560 565 570 Val Met Ser Val Ala Asn Ile Pro Leu Ser Met Pro Glu Leu Leu 575 580 585 Val Glu Val Pro Gly Ser Ile Ile Val Gln Glu Lys Val Thr Ala 590 595 600 Phe Ile Ser Phe Thr Asn Pro Leu Thr Val Pro Leu Lys Arg Gly 605 610 615 Met Phe Thr Val Glu Gly Ser Gly Leu Leu Ser Ala Ser Glu Ile 620 625 630 Tyr Val Lys Gly Asp Ile Ala Pro Gly Gln Lys Val Ser Val Lys 635 640 645 Ile Thr Phe Thr Pro Met Arg Val Gly Val Arg Lys Leu Leu Val 650 655 660 Asp Phe Asp Ser Asp Arg Leu Lys Asp Val Lys Gly Val Thr Thr 665 670 675 Val Val Val Arg Lys Lys Ser Cys Phe Ile Arg Cys Pro 680 685 SEQ ID NO: 45 Sequence Length: 2054 Sequence Type: Nucleic Acid Strand Count: Double-Strand Topology Type of linear sequence: cDNA to mRNA Origin organism name: Flounder Character of liver sequence Characteristic symbol: CDS Location: 1..2054 Method of determining feature: P sequence GACAATCAGA ACATTCCGAT CACTGATGTG GATGTGAGAA GTCATGAAAA CAACTTGGCT 60 CACCGCACCA GGGAGATTGA TCGGGAGC TTGATCGTCC GCAGGGGTCA ACCCTTCTCC 120 ATATCTCTGC AGTGCTGCGA CTCGCTGACC CGGAATCACC ATCTGGAACT GTCCCTGCAC 180 CTCGGTAAGA AAGATGAGGT GGTGATTAAG GTGCACAATG AGCCTGAGGC TGGAGGCAAG 240 TGGTGGTTTA ACCATCAGAA AGTGCAGGAT GAAATTCTGC TGACTCTACA CAGTCCAGCG 300 GACGCCATAA TTGGCGAGTA CCACCTGACT GTGTTGATCA AGTCACCGGA TGGACACTTT 360 GTGAAGAAGA CTAAGAACAT TGGATTCCAC CTGCTCTTTA ACCCCTGGTG CAAAGATGAT 420 GCTGTGTACC TCCCTGATGA AAGGATGCTC GACGAGTATG TTATGAATGA GGAGGGGATC 480 ATTTACAGGG GAACCTCGAA TCACATCAGT AGCATACCCT GGAATTACGG ACAGTTTGAG 540 GACTATGTGA TGGACATCTG TTTTCAAGTT CTGGACAACT CCAAGGAAGC CCTGAAGAAT 600 TCAAAGATGG ACATTGAGAA GAGATCTGAC CCTGTCTATG TCAGCAGGAT GATCACTGCG 660 ATGGTGAACT CTAACGGTGA CAGGGGTGTG CTGACTGGTCGG ACGA GCCATACACT 720 GGCGGGTTCT CACCACTTCG ATGGACCGGC AGCGTGCCCA TCCTCCGGAA GTGGAGCAAG 780 GCCGAGGTCA GGGCGGTCAA ATATGGCCAG TGCTGGGTGT TTGCTGCTGT CGCCTGCACA 840 GTGCTGCGTT GTCTGGGAAT CCCAACACGC AACATCACTA ACTTCAATTC AGCACATGAT 900 GTCGATGGAA ACCTCTCCGT CGACATCGTG TTGAACAAAG AAATGGAGAG CGTTGGCAAG 960 AAGGACAGTA GCTGGAACTT CCACTGTTGG ATCGAGTCCT GGATGAGGAG AGACGACCTC 1020 TCTAAAGGAA ATGACGGCTG GCAGGTTTTG GACCCCACCC CTCAAGAACT GAGTGATGGT 1080 GAGTATTGCT GCGGCCCGTG TCCAGTCACC GCCATCAAGG AGGGAAATCT GAGTGTGAAG 1140 TACGACGCTC CGTTTATCTT CGCTGAGGTG AACGCTGACA TCATCTACTG GATGGCTGGA 1200 CCAGGAGGCG AACGGAAGAA GATCGATGTG GACCAGAGTG GTGTGGGGAA GAACATCAGC 1260 ACCAAAAGTC TTTATGGCGA CTACAGGGAG GATGTCACTC TGCACTACAA ATACCCCGAA 1320 GGCTCCAAGA AGGAGAGAGA GGTGTACCAG AAGGCCGGAC ACCGAATCAA AGAGCAGATC 1380 TGTGAAAACA AAGGTCCACA ACAACTGCAG CTGTCAGTCA AGCACGGGAA ACCTGTATTT 1440 GGCACTGACT TCGATGTGAT AGTTGAGGTG AAGAATGAAG GACAGAAAGA CACCAGTCCA 1500 CAGCTGCTGA TTGTGGTCAT GGCCGTGACC TACAATTCCA TCAATCAAGG GGAGTG TCAG 1560 AGGAAGGCGA CCATAGTGAC CGTGCCGGCT CGCAAAACCC ACAAGGAAGT GCTGCGTCTG 1620 CGCTACGACG ACTATGTCAA ATGTGTCTCT GAGCACCATC TGATCAGGGT GAAAGCGCTC 1680 ATGGAGGTTC CAGGGGACAA CAAACCCGTC ATGAGTGTGG CCAACATTCC ACTGAGCATG 1740 CCTGAGCTCC TGGTAGAGGT ACCTGGGAGC ATCATTGTTC AGGAGAAGGT GACAGCCTTC 1800 ATCTCCTTCA CAAATCCTCT AACTGTCCCA CTGAAGCGTG GCATGTTCAC CGTTGAGGGG 1860 TCCGGACTAC TGTCTGCCTC TGAGATCTAT GTGAAAGGGG ACATTGCTCC AGGCCAGAAG 1920 GTTTCTGTCA AGATCACCTT CACGCCCATG AGGGTCGGGG TGAGGAAGCT CCTGGTGGAC 1980 TTTGACTCTG ACAGGCTGAA GGATGTGAAA GGAGTCACGA CAGTGGTCGT CCGCAAGAAA 2040 TCCTGTTTTA TTAGGTGTCC TTAA 2054 SEQ ID NO: 46 Sequence length: 2148 Sequence type: Nucleic acid chain number: Double-stranded topology: Linear array type: cDNA to mRNA Origin organism name: Flounder Character symbol of liver sequence: CDS Location: 26..2092 Method of determining feature: P sequence GAGAAGACGA GGAAAAAGGT CTGCG ATG GAC AAT CAG AAC ATT CCG ATC ACT 52 Met Asp Asn Gln Asn Ile Pro Ile Thr 1 5 G AT GTG GAT GTG AGA AGT CAT GAA AAC AAC TTG GCT CAC CGC ACC 97 Asp Val Asp Val Arg Ser His Glu Asn Asn Leu Ala His Arg Thr 10 15 20 AGG GAG ATT GAT CGG GAG CGC TTG ATC GTC CGC AGG GGT CAA CCC 142 Arg Glu Ile Asp Arg Glu Arg Leu Ile Val Arg Arg Gly Gln Pro 25 30 35 TTC TCC ATA TCT CTG CAG TGC TGC GAC TCG CTG ACC CGG AAT CAC 187 Phe Ser Ile Ser Leu Gln Cys Cys Asp Ser Leu Thr Arg Asn His 40 45 50 CAT CTG GAA CTG TCC CTG CAC CTC GGT AAG AAA GAT GAG GTG GTG 232 His Leu Glu Leu Ser Leu His Leu Gly Lys Lys Asp Glu Val Val 55 60 65 ATT AAG GTG CAC AAT GAG CCT GAG GCT GGA GGC AAG TGG TGG TTT 277 Ile Lys Val His Asn Glu Pro Glu Ala Gly Gly Lys Trp Trp Phe 70 75 80 AAC CAT CAG AAA GTG CAG GAT GAA ATT CTG CTG ACT CTA CAC AGT 322 Asn His Gln Lys Val Gln Asp Glu Ile Leu Leu Thr Leu His Ser 85 90 95 CCA GCG GAC GCC ATA ATT GGC GAG TAC CAC CTG ACT GTG TTG ATC 367 Pro Ala Asp Ala Ile Ile Gly Glu Tyr His Leu Thr Val Leu Ile 100 105 110 AAG TCA CCG GAT GGA CAC TTT GTG AAG AAG ACT AAG AAC ATT GGA 412 Lys Ser Pro Asp Gly His Phe Val Lys Lys Thr Lys Asn Ile Gly 115 120 125 TTC CAC CTG CTC TTT AAC CCC TGG TGC AAA GAT GAT GCT GTG TAC 457 Phe His Leu Leu Phe Asn Pro Trp Cys Lys Asp Asp Ala Val Tyr 130 135 140 CTC CCT GAT GAA AGG ATG CTC GAC GAG TAT GTT ATG AAT GAG GAG 502 Leu Pro Asp Glu Arg Met Leu Asp Glu Tyr Val Met Asn Glu Glu 145 150 155 GGG ATC ATT TAC AGG GGA ACC TCG AAT CAC ATC AGT AGC ATA CCC 547 Gly Ile Ile Tyr Arg Gly Thr Ser Asn His Ile Ser Ser Ile Pro 160 165 170 TGG AAT TAC GGA CAG TTT GAG GAC TAT GTG ATG GAC ATC TGT TTT 592 Trp Asn Tyr Gly Gln Phe Glu Asp Tyr Val Met Asp Ile Cys Phe 175 180 185 CAA GTT CTG GAC AAC TCC AAG GAA GCC CTG AAG AAT TCA AAG ATG 637 Gln Val Leu Asp Asn Ser Lys Glu Ala Leu Lys Asn Ser Lys Met 190 195 200 GAC ATT GAG AAG AGA TCT GAC CCT GTC TAT GTC AGC AGG ATG ATC 682 Asp Ile Glu Lys Arg Ser Asp Pro Val Tyr Val Ser Arg Met Ile 205 210 215 ACT GCG ATG GTG AAC TCT AAC GGT GAC AGG GGT GTG CTG ACT GGT 727 Thr Ala Met Val Asn Ser Asn Gly Asp Arg Gly Val Leu Thr Gly 220 225 230 CAG TGG CAC GAG CCA TAC ACT GGC GGG TTC TCA CCA CTT CGA TGG 772 Gln Trp His Glu Pro Tyr Thr Gly Gly Phe Ser Pro Leu Arg Trp 235 240 245 ACC GGC AGC GTG CCC ATC CTC CGG AAG TGG AGC AAG GCC GAG GTC 817 Thr Gly Ser Val Pro Ile Leu Arg Lys Trp Ser Lys Ala Glu Val 250 255 260 AGG GCG GTC AAA TAT GGC CAG TGC TGG GTG TTT GCT GCT GTC GCC 862 Arg Ala Val Lys Tyr Gly Gln Cys Trp Val Phe Ala Ala Val Ala 265 270 275 TGC ACA GTG CTG CGT TGT CTG GGA ATC CCA ACA CGC AAC ATC ACT 907 Cys Thr Val Leu Arg Cys Leu Gly Ile Pro Thr Arg Asn Ile Thr 280 285 290 AAC TTC AAT TCA GCA CAT GAT GTC GAT GGA AAC CTC TCC GTC GAC 952 Asn Phe Asn Ser Ala His Asp Val Asp Gly Asn Leu Ser Val Asp 295 300 305 ATC GTG TTG AAC AAA GAA ATG GAG AGC GTT GGC AAG AAG GAC AGT 997 Ile Val Leu Asn Lys Glu Met Glu Ser Val Gly Lys Lys Asp Ser 310 315 320 AGC TGG AAC TTC CAC TGT TGG ATC GAG TCC TGG ATG AGG AGA GAC 1042 Ser Trp Asn Phe His Cys Trp Ile Glu Ser Trp Met Arg Arg Asp 325 330 335 GAC CTC TCT AAA GGA AAT GAC GGC TGG CAG GTT TTG GAC CCC ACC 1087 Asp Leu Ser Lys Gly Asn Asp Gly Trp Gln Val Leu Asp Pro Thr 340 345 350 CCT CAA GAA CTG AGT GAT GGT GAG TAT TGC TGC GGC CCG TGT CCA 1132 Pro Gln Glu Leu Ser Asp Gly Glu Tyr Cys Cys Gly Pro Cys Pro 355 360 365 GTC ACC GCC ATC AAG GAG GGA AAT CTG AGT GTG AAG TAC GAC GCT 1177 Val Thr Ala Ile Lys Glu Gly Asn Leu Ser Val Lys Tyr Asp Ala 370 375 380 CCG TTT ATC TTC GCT GAG GTG AAC GCT GAC ATC ATC TAC TGG ATG 1222 Pro Phe Ile Phe Ala Glu Val Asn Ala Asp Ile Ile Tyr Trp Met 385 390 395 GCT GGA CCA GGA GGC GAA CGG AAG AAG ATC GAT GTG GAC CAG AGT 1267 Ala Gly Pro Gly Gly Glu Arg Lys Lys Ile Asp Val Asp Gln Ser 400 405 410 GGT GTG GGG AAG AAC ATC AGC ACC AAA AGT CTT TAT GGC GAC TAC 1312 Gly Val Gly Lys Asn Ile Ser Thr Lys Ser Leu Tyr Gly Asp Tyr 415 420 425 AGG GAG GAT GTC ACT CTG CAC TAC AAA TAC CCC GAA GGC TCC AAG 1357 Arg Glu Asp Val Thr Leu His Tyr Lys Tyr Pro Glu Gly Ser Lys 430 435 440 AAG GAG AGA GAG GTG TAC CAG AAG GCC GGA CAC CGA ATC AAA GAG 1402 Lys Glu Arg Glu Val Tyr Gln Lys Ala Gly His Arg Ile Lys Glu 445 450 455 CAG ATC TGT GAA AAC AAA GGT CCA CAA CAA CTG CAG CTG TCA GTC 1447 Gln Ile Cys Glu Asn Lys Gly Pro Gln Gln Leu Gln Leu Ser Val 460 465 470 AAG CAC GGG AAA CCT GTA TTT GGC ACT GAC TTC GAT GTG ATA GTT 1492 Lys His Gly Lys Pro Val Phe Gly Thr Asp Phe Asp Val Ile Val 475 480 485 GAG GTG AAG AAT GAA GGA CAG AAA GAC ACC AGT CCA CAG CTG CTG 1537 Glu Val Lys Asn Glu Gly Gln Lys Asp Thr Ser Pro Gln Leu Leu 490 495 500 ATT GTG GTC ATG GCC GTG ACC TAC AAT TCC ATC AAT CAA GGG GAG 1582 Ile Val Val Met Ala Val Thr Tyr Asn Ser Ile Asn Gln Gly Glu 505 510 515 TGT CAG AGG AAG GCG ACC ATA GTG ACC GTG CCG GCT CGC AAA ACC 1627 Cys Gln Arg Lys Ala Thr Ile Val Thr Val Pro Ala Arg Lys Thr 520 525 530 CAC AAG GAA GTG CTG CGT CTG CGC TAC GAC GAC TAT GTC AAA TGT 1672 His Lys Glu Val Leu Arg Leu Arg Tyr Asp Asp Tyr Val Lys Cys 535 540 545 GTC TCT GAG CAC CAT CTG ATC AGG GTG AAA GCG CTC ATG GAG GTT 1717 Val Ser Glu His His Leu Ile Arg Val Lys Ala Leu Met Glu Val 550 555 560 CCA GGG GAC AAC AAA CCC GTC ATG AGT GTG GCC AAC ATT CCA CTG 1762 Pro Gly Asp Asn Lys Pro Val Met Ser Val Ala Asn Ile Pro Leu 565 570 575 AGC ATG CCT GAG CTC CTG GTA GAG GTA CCT GGG AGC ATC ATT GTT 1807 Ser Met Pro Glu Leu Leu Val Glu Val Pro Gly Ser Ile Ile Val 580 585 590 CAG GAG AAG GTG ACA GCC TTC ATC TCC TTC ACA AAT CCT CTA ACT 1852 Gln Glu Lys Val Thr Ala Phe Ile Ser Phe Thr Asn Pro Leu Thr 595 600 605 GTC CCA CTG AAG CGT GGC ATG TTC ACC GTT GAG GGG TCC GGA CTA 1897 Val Pro Leu Lys Arg Gly Met Phe Thr Val Glu Gly Ser Gly Leu 610 615 620 CTG TCT GCC TCT GAG ATC TAT GTG AAA GGG GAC ATT GCT CCA GGC 1942 Leu Ser Ala Ser Glu Ile Tyr Val Lys Gly Asp Ile Ala Pro Gly 625 630 635 CAG AAG GTT TCT GTC AAG ATC ACC TTC ACG CCC ATG AGG GTC GGG 1987 Gln Lys Val Ser Val Lys Ile Thr Phe Thr Pro Met Arg Val Gly 640 645 650 GTG AGG AAG CTC CTG GTG GAC TTT GAC TCT GAC AGG CTG AAG GAT 2032 Val Arg Lys Leu Leu Val Asp Phe Asp Ser Asp Arg Leu Lys Asp 655 660 665 GTG AAA GGA G TC ACG ACA GTG GTC GTC CGC AAG AAA TCC TGT TTT 2077 Val Lys Gly Val Thr Thr Val Val Val Arg Lys Lys Ser Cys Phe 670 675 680 ATT AGG TGT CCT TAAAAACAGA CGGACACGTA TTAAAGTGTG AGATAACCTG 2129 Ile Arg Cys Pro 685 TCAGGTGTAA : 47 Sequence length: 2054 Sequence type: Nucleic acid chain number: Double-stranded topology: Linear sequence type: cDNA to mRNA origin Organism name: flounder Symbol representing characteristic features of liver sequence: CDS Location: 1..2054 method to determine the characteristics: P sequence GACAATCAGA ACATTCCGAT CACTGATGTG GATGTGAGAA GTCATGAAAA CAACTTGGCT 60 CACCGCACCA GGGAGATTGA TCGGGAGCGC TTGATCGTCC GCAGGGGTCA ACCCTTCTCC 120 ATATCTCTGC AGTGCTGCGA CTCGCTGACC CGGAATCACC ATCTGGAACT GTCCCTGCAC 180 CTCGGTAAGA AAGATGAGGT GGTGATTAAG GTGCACAATG AGCCTGAGGC TGGAGGCAAG 240 TGGTGGTTTA ACCATCAGAA AGTGCAGGAT GAAATTCTGC TGACTCTACA CAGTCCAGCG 300 GACGCCATAA TTGGCGAGTA CCACCTGACT GTGTTGATCA AGTCACCGGA TGGACACTTT 360 GTGAAGAAGA CTAAGAACAT TGGATTCCAC CTGCTCTTTA ACCCCTGGTG CAAAGATGAT 420 GCTGTGTACC TCCCTGATGA AAGGATGCTC GACGAGTATG TTATGAATGA GGAGGGGATC 480 ATTTACAGGG GAACCTCGAA TCACATCAGT AGCATACCCT GGAATTACGG ACAGTTTGAG 540 GACTATGTGA TGGACATCTG TTTTCAAGTT CTGGACAACT CCAAGGAAGC CCTGAAGAAT 600 TCAAAGATGG ACATTGAGAA GAGATCTGAC CCTGTCTATG TCAGCAGGAT GATCACTGCG 660 ATGGTGAACT CTAACGGTGA CAGGGGTGTG CTGACTGGTC AGTGGCACGA GCCATACACT 720 GGCGGGTTCT CACCACTTCG ATGGACCGGC AGCGTGCCCA TCCTCCGGAA GTGGAGCAAG 780 GCCGAGGTCA GGGCGGTCAA ATATGGCCAG TGCTGGGTGT TTGCTGCTGT CGCCTGCACA 840 GTGCTGCGTT GTCTGGGAAT CCCAACACGC AACATCACTA ACTTCAATTC AGCACATGAT 900 GTCGATGGAA ACCTCTCCGT CGACATCGTG TTGAACAAAG AAATGGAGAG CGTTGGCAAG 960 AAGGACAGTA GCTGGAACTT CCACTGTTGG ATCGAGTCCT GGATGAGGAG AGACGACCTC 1020 TCTAAAGGAA ATGACGGCTG GCAGGTTTTG GACCCCACCC CTCAAGAACT GAGTGATGGT 1080 GAGTATTGCT GCGGCCCGTG TCCAGTCACC GCCATCAAGG AGGGAAATCT GAGTGTGAAG 1140 TACGACGCTC CGTTTATCTT CGCTGAGGTG AACGCTGACA TCATCTACTG GATGGCTGGA 1200 CCAGGAGGCG AACGGAAGAA GATCGATGTG GACCAGAGTG GTGTGGGGAA GAACATCAGC 1260 ACCAAAAGTC TTTATGGCGA CTACAGGGAG GATGTCACTC TGCACTACAA ATACCCCGAA 1320 GGCTCCAAGA AGGAGAGAGA GGTGTACCAG AAGGCCGGAC ACCGAATCAA AGAGCAGATC 1380 TGTGAAAACA AAGGTCCACA ACAACTGCAG CTGTCAGTCA AGCACGGGAA ACCTGTATTT 1440 GGCACTGACT TCGATGTGAT AGTTGAGGTG AAGAATGAAG GACAGAAAGA CACCAGTCCA 1500 CAGCTGCTGA TTGTGGTCAT GGCCGTGACC TACAATTCCA TCAATCAAGG GGAGTGTCAG 1560 AGGAAGGCGA CCATAGTGAC CGTGCCGGCT CGCAAAACCC ACAAGGAAGT GCTGCGTCTG 1620 CGCTACGACG ACTATGTCAA ATGTGTCTCT GAGCACCATC TGATCAGGGT GAAAGCGCTC 1680 ATGGAGGTTC CAGGGGACAA CAAACCCGTC ATGAGTGTGG CCAACATTCC ACTGAGCATG 1740 CCTGAGCTCC TGGTAGAGGT ACCTGGGAGC ATCATTGTTC AGGAGAAGGT GACAGCCTTC 1800 ATCTCCTTCA CAAATCCTCT AACTGTCCCA CTGAAGCGTG GCATGTTCAC CGTGGAGGGG 1860 TCCGGACTAC TGTCTGCCTC TGAGATCTAT GTGAAAGGGG ACATTGCTCC AGGCCAGAAG 1920 GTTTCTGTCA AGATCACCTT CACGCCCATG AGGGTCGGGG TGAGGAAGCT CCTGGTGGAC 1980 TTTGACTCTG ACAGGCTGAA GGATGTGAAA GGAGTCACGA CAGTGGTCGT CCGCAAGAAA 2040 TCCTGTTTTA TTAGGTGTCC TTAA 2054 SEQ ID NO: 48 array of Length: 2148 Array type Number of nucleic acid strands: Double-stranded topology: Type of linear sequence: cDNA to mRNA origin Organism name: flounder Liver sequence features Characteristic symbols: CDS Location: 26..2092 Method of determining features: P sequence GAGAAGACGA GGAAAAAGGT CTGCG ATG GAC AAT CAG AAC ATT CCG ATC ACT 52 Met Asp Asn Gln Asn Ile Pro Ile Thr 1 5 GAT GTG GAT GTG AGA AGT CAT GAA AAC AAC TTG GCT CAC CGC ACC 97 Asp Val Asp Val Arg Ser His Glu Asn As Leu Ala His Arg Thr 10 15 20 AGG GAG ATT GAT CGG GAG CGC TTG ATC GTC CGC AGG GGT CAA CCC 142 Arg Glu Ile Asp Arg Glu Arg Leu Ile Val Arg Arg Gly Gln Pro 25 30 35 TTC TCC ATA TCT CTG CAG TGC TGC GAC TCG CTG ACC CGG AAT CAC 187 Phe Ser Ile Ser Leu Gln Cys Cys Asp Ser Leu Thr Arg Asn His 40 45 50 CAT CTG GAA CTG TCC CTG CAC CTC GGT AAG AAA GAT GAG GTG GTG 232 His Leu Glu Leu Ser Leu His Leu Gly Lys Lys Asp Glu Val Val 55 60 65 ATT AAG GTG CAC AAT GAG CCT GAG GCT GGA GGC AAG TGG TGG TTT 277 Ile Lys Val His Asn Glu Pro Glu Ala Gly Gly Lys Trp Trp Phe 70 75 80 AA C CAT CAG AAA GTG CAG GAT GAA ATT CTG CTG ACT CTA CAC AGT 322 Asn His Gln Lys Val Gln Asp Glu Ile Leu Leu Thr Leu His Ser 85 90 95 CCA GCG GAC GCC ATA ATT GGC GAG TAC CAC CTG ACT GTG TTG ATC 367 Pro Ala Asp Ala Ile Ile Gly Glu Tyr His Leu Thr Val Leu Ile 100 105 110 AAG TCA CCG GAT GGA CAC TTT GTG AAG AAG ACT AAG AAC ATT GGA 412 Lys Ser Pro Asp Gly His Phe Val Lys Lys Thr Lys Asn Ile Gly 115 120 125 TTC CAC CTG CTC TTT AAC CCC TGG TGC AAA GAT GAT GCT GTG TAC 457 Phe His Leu Leu Phe Asn Pro Trp Cys Lys Asp Asp Ala Val Tyr 130 135 140 CTC CCT GAT GAA AGG ATG CTC GAC GAG TAT GTT ATG AAT GAG GAG 502 Leu Pro Asp Glu Arg Met Leu Asp Glu Tyr Val Met Asn Glu Glu 145 150 155 GGG ATC ATT TAC AGG GGA ACC TCG AAT CAC ATC AGT AGC ATA CCC 547 Gly Ile Ile Tyr Arg Gly Thr Ser Asn His Ile Ser Ser Ile Pro 160 165 170 TGG AAT TAC GGA CAG TTT GAG GAC TAT GTG ATG GAC ATC TGT TTT 592 Trp Asn Tyr Gly Gln Phe Glu Asp Tyr Val Met Asp Ile Cys Phe 175 180 185 CAA GTT CTG GAC AAC TCC AAG GAA GCC CTG AAG AAT TCA A AG ATG 637 Gln Val Leu Asp Asn Ser Lys Glu Ala Leu Lys Asn Ser Lys Met 190 195 200 GAC ATT GAG AAG AGA TCT GAC CCT GTC TAT GTC AGC AGG ATG ATC 682 Asp Ile Glu Lys Arg Ser Asp Pro Val Tyr Val Ser Arg Met Ile 205 210 215 ACT GCG ATG GTG AAC TCT AAC GGT GAC AGG GGT GTG CTG ACT GGT 727 Thr Ala Met Val Asn Ser Asn Gly Asp Arg Gly Val Leu Thr Gly 220 225 230 CAG TGG CAC GAG CCA TAC ACT GGC GGG TTC TCA CCA CTT CGA TGG 772 Gln Trp His Glu Pro Tyr Thr Gly Gly Phe Ser Pro Leu Arg Trp 235 240 245 ACC GGC AGC GTG CCC ATC CTC CGG AAG TGG AGC AAG GCC GAG GTC 817 Thr Gly Ser Val Pro Ile Leu Arg Lys Trp Ser Lys Ala Glu Val 250 255 260 AGG GCG GTC AAA TAT GGC CAG TGC TGG GTG TTT GCT GCT GTC GCC 862 Arg Ala Val Lys Tyr Gly Gln Cys Trp Val Phe Ala Ala Val Ala 265 270 275 TGC ACA GTG CTG CGT TGT CTG GGA ATC CCA ACA CGC AAC ATC ACT 907 Cys Thr Val Leu Arg Cys Leu Gly Ile Pro Thr Arg Asn Ile Thr 280 285 290 AAC TTC AAT TCA GCA CAT GAT GTC GAT GGA AAC CTC TCC GTC GAC 952 Asn Phe Asn Ser Ala His Asp Val Asp G ly Asn Leu Ser Val Asp 295 300 305 ATC GTG TTG AAC AAA GAA ATG GAG AGC GTT GGC AAG AAG GAC AGT 997 Ile Val Leu Asn Lys Glu Met Glu Ser Val Gly Lys Lys Asp Ser 310 315 320 AGC TGG AAC TTC CAC TGT TGG ATC GAG TCC TGG ATG AGG AGA GAC 1042 Ser Trp Asn Phe His Cys Trp Ile Glu Ser Trp Met Arg Arg Asp 325 330 335 GAC CTC TCT AAA GGA AAT GAC GGC TGG CAG GTT TTG GAC CCC ACC 1087 Asp Leu Ser Lys Gly Asn Asp Gly Trp Gln Val Leu Asp Pro Thr 340 345 350 CCT CAA GAA CTG AGT GAT GGT GAG TAT TGC TGC GGC CCG TGT CCA 1132 Pro Gln Glu Leu Ser Asp Gly Glu Tyr Cys Cys Gly Pro Cys Pro 355 360 365 GTC ACC GCC ATC AAG GAG GGA AAT CTG AGT GTG AAG TAC GAC GCT 1177 Val Thr Ala Ile Lys Glu Gly Asn Leu Ser Val Lys Tyr Asp Ala 370 375 380 CCG TTT ATC TTC GCT GAG GTG AAC GCT GAC ATC ATC TAC TGG ATG 1222 Pro Phe Ile Phe Ala Glu Val Asn Ala Asp Ile Ile Tyr Trp Met 385 390 395 GCT GGA CCA GGA GGC GAA CGG AAG AAG ATC GAT GTG GAC CAG AGT 1267 Ala Gly Pro Gly Gly Glu Arg Lys Lys Ile Asp Val Asp Gln Ser 400 405 410 GGT GTG GGG AAG AAC ATC AGC ACC AAA AGT CTT TAT GGC GAC TAC 1312 Gly Val Gly Lys Asn Ile Ser Thr Lys Ser Leu Tyr Gly Asp Tyr 415 420 425 AGG GAG GAT GTC ACT CTG CAC TAC AAA TAC CCC GAA GGC TCC AAG 1357 Arg Glu Asp Val Thr Leu His Tyr Lys Tyr Pro Glu Gly Ser Lys 430 435 440 AAG GAG AGA GAG GTG TAC CAG AAG GCC GGA CAC CGA ATC AAA GAG 1402 Lys Glu Arg Glu Val Tyr Gln Lys Ala Gly His Arg Ile Lys Glu 445 450 455 CAG ATC TGT GAA AAC AAA GGT CCA CAA CAA CTG CAG CTG TCA GTC 1447 Gln Ile Cys Glu Asn Lys Gly Pro Gln Gln Leu Gln Leu Ser Val 460 465 470 AAG CAC GGG AAA CCT GTA TTT GGC ACT GAC TTC GAT GTG ATA GTT 1492 Lys His Gly Lys Pro Val Phe Gly Thr Asp Phe Asp Val Ile Val 475 480 485 GAG GTG AAG AAT GAA GGA CAG AAA GAC ACC AGT CCA CAG CTG CTG 1537 Glu Val Lys Asn Glu Gly Gln Lys Asp Thr Ser Pro Gln Leu Leu 490 495 500 ATT GTG GTC ATG GCC GTG ACC TAC AAT TCC ATC AAT CAA GGG GAG 1582 Ile Val Val Met Ala Val Thr Tyr Asn Ser Ile Asn Gln Gly Glu 505 510 515 TGT CAG AGG AAG GCG ACC ATA GTG ACC GTG CCG GCT CGC AAA ACC 1627 Cys Gln Arg Lys Ala Thr Ile Val Thr Val Pro Ala Arg Lys Thr 520 525 530 CAC AAG GAA GTG CTG CGT CTG CGC TAC GAC GAC TAT GTC AAA TGT 1672 His Lys Glu Val Leu Arg Leu Arg Tyr Asp Asp Tyr Val Lys Cys 535 540 545 GTC TCT GAG CAC CAT CTG ATC AGG GTG AAA GCG CTC ATG GAG GTT 1717 Val Ser Glu His His Leu Ile Arg Val Lys Ala Leu Met Glu Val 550 555 560 CCA GGG GAC AAC AAA CCC GTC ATG AGT GTG GCC AAC ATT CCA CTG 1762 Pro Gly Asp Asn Lys Pro Val Met Ser Val Ala Asn Ile Pro Leu 565 570 575 AGC ATG CCT GAG CTC CTG GTA GAG GTA CCT GGG AGC ATC ATT GTT 1807 Ser Met Pro Glu Leu Leu Val Glu Val Pro Gly Ser Ile Ile Val 580 585 590 CAG GAG AAG GTG ACA GCC TTC ATC TCC TTC ACA AAT CCT CTA ACT 1852 Gln Glu Lys Val Thr Ala Phe Ile Ser Phe Thr Asn Pro Leu Thr 595 600 605 GTC CCA CTG AAG CGT GGC ATG TTC ACC GTG GAG GGG TCC GGA CTA 1897 Val Pro Leu Lys Arg Gly Met Phe Thr Val Glu Gly Ser Gly Leu 610 615 620 CTG TCT GCC TCT GAG ATC TAT GTG AAA GGG GAC ATT GCT CCA GGC 1942 Leu Ser Ala Ser Glu Ile Tyr Val Lys Gly Asp Ile Ala Pro Gly 625 630 635 CAG AAG GTT TCT GTC AAG ATC ACC TTC ACG CCC ATG AGG GTC GGG 1987 Gln Lys Val Ser Val Lys Ile Thr Phe Thr Pro Met Arg Val Gly 640 645 650 GTG AGG AAG CTC CTG GTG GAC TTT GAC TCT GAC AGG CTG AAG GAT 2032 Val Arg Lys Leu Leu Val Asp Phe Asp Ser Asp Arg Leu Lys Asp 655 660 665 GTG AAA GGA GTC ACG ACA GTG GTC GTC CGC AAG AAA TCC TGT TTT 2077 Val Lys Gly Val Thr Thr Val Val Val Arg Lys Lys Ser Cys Phe 670 675 680 ATT AGG TGT CCT TAAAAACAGA CGGACACGTA TTAAAGTGTG AGATAACCTG 2129 Ile Arg Cys Pro 685 AGAGGTGTAA CTCCCCTGT 2148

SEQ ID NO: 49 Sequence Length: 640 Sequence Type: Amino Acid Topology: Linear Sequence Type: Protein Origin Biological Name: Pollack Muscle Name Sequence Ala His Thr Asn Arg Leu Ile Ala Gly Val Asp Leu Arg Ser Gln 1 5 10 15 Glu Asn Asn Arg Glu His Arg Thr Glu Glu Ile Asp Arg Lys Arg 20 25 30 Leu Ile Val Arg Arg Gly Gln Ala Phe Ser Leu Thr Val His Leu 35 40 45 Ser Asp Pro Leu Gln Ser Gly His Glu Leu Ala Leu Val Leu Lys 50 55 60 Gln Asp Lys Ile Asn Asp Asp Ile Val Ile Arg Gln Arg Thr Thr 65 70 75 Gly Gly Ser Gly Asp Lys Trp Trp Leu His Gln Gln Ser Ala Asn 80 85 90 Asn Glu Leu Leu Leu Thr Val Tyr Ser Pro Ala Arg Ala Ala Val 95 100 105 Gly Glu Tyr Arg Leu Ala Val Glu Leu Met Ser Gly Asn Lys Leu 110 115 120 Leu Glu Arg Thr Asp Phe Thr Lys Met Tyr Leu Leu Phe Asn Pro 125 130 135 Trp Cys Lys Glu Asp Ala Val Tyr Leu Pro Asp Glu Cys Leu Leu 140 145 150 Lys Glu Tyr Ile Met Asn Glu Asn Gly Arg Ile Phe Thr Gly Ser 155 160 165 Ala Asp Trp Met Ser Gly Leu Pr o Trp Asn Phe Gly Gln Phe Glu 170 175 180 Asp Asn Val Met Asp Ile Cys Phe Glu Ile Leu Asp Arg Phe Asn 185 190 195 Pro Ala Arg Ser Asp Pro Pro Ser Asp Met Leu Gln Arg Trp Asp 200 205 210 Pro Val Tyr Ile Ser Arg Ala Val Val Ala Met Val Asn Ala Asn 215 220 225 Asp Asp Asp Gly Gly Val Val Val Gly Arg Trp Gln Glu Pro Tyr 230 235 240 Thr Gly Gly Val Gln Pro Thr Lys Trp Met Ser Ser Val Pro Ile 245 250 255 Leu Glu Glu Trp Ser Lys Ser Lys Ser Gly Val Lys Tyr Gly Gln 260 265 270 Cys Trp Val Phe Ala Ala Val Ala Cys Thr Val Met Arg Cys Leu 275 280 285 Gly Ile Pro Thr Arg Cys Ile Thr Asn Phe Gln Ser Ala His Asp 290 295 300 Thr Asp Gly Asn Leu Ser Ile Asp Arg Val Tyr Asn Ile His Arg 305 310 315 Gln Leu Val Asp Gly Asp Asp Ser Ile Trp Asn Phe His Cys Trp 320 325 330 Ile Glu Ser Tyr Met Gln Arg Glu Asp Leu Pro Glu Gly Tyr Gly 335 340 345 Gly Trp Gln Val Leu Asp Pro Thr Pro Gln Glu Arg Ser Ser Gly 350 355 360 Met Phe Arg Cys Gly Pro Cys Pro Leu Lys Ala Ile Lys Glu Gly 365 370 375 Asp Leu Asn Val Lys Ph e Asp Val Pro Phe Val Phe Ala Glu Val 380 385 390 Asn Ala Asp Ile Ile Asn Trp Glu Ile Arg Pro Asp Gly Gln Arg 395 400 405 Lys Arg Leu Ser Ser Asn Ser Ala Asn Val Gly Arg Asn Ile Ser 410 415 420 Thr Lys Ser Pro Tyr Gly Asn Glu Arg Glu Asp Ile Thr His Gln 425 430 435 Tyr Lys Tyr Gln Glu Gly Ser Ala Lys Glu Arg Glu Val Tyr Asn 440 445 450 Lys Ala Gly Arg Arg Ile Ser Gly Pro Asp Gly Glu Glu Glu Ser 455 460 465 Lys Pro Gly Asn Val Gln Leu Glu Ile Lys His Ala Lys Pro Val 470 475 480 Phe Gly Thr Asp Phe Asp Val Ile Phe Glu Leu Glu Asn Met Gly 485 490 495 Asp Lys Glu Val Ser Cys Lys Leu Asn Met Met Ser Glu Ala Val 500 505 510 Thr Tyr Asn Ser Val His Leu Gly Arg Phe Gln Asn Ser Thr Val 515 520 525 Asn Val Val Ile Pro Ala His Lys Val His Ser Glu Thr Val Arg 530 535 540 Leu Leu Tyr Thr Lys Tyr Ala Ser Val Val Ser Glu His Asn Ile 545 550 555 Ile Arg Val Thr Gly Val Ala Glu Val Ser Gly Gln Glu Lys Ser 560 565 570 Ile Leu Glu Met Val Asn Ile Pro Leu Ser Lys Pro Lys Leu Ser 575 580 585 Ile Lys Val Pr o Gly Trp Val Ile Leu Asn Arg Lys Ile Thr Thr 590 595 600 Phe Ile Ser Phe Thr Asn Pro Leu Pro Val Pro Leu Asn Arg Gly 605 610 615 Val Phe Thr Val Glu Gly Ala Gly Leu Leu Pro Thr Lys Glu Ile 620 625 630 Arg Ile Ser Gly Ser Ile Ala Pro Gly Gln 635 640 SEQ ID NO: 50 Sequence Length: 641 Sequence Type: Amino Acid Topology: Linear Sequence Type: Protein Origin Biological Name: Pollack Cod Muscle Sequence Met Ala His Thr Asn Arg Leu Ile Ala Gly Val Asp Leu Arg Ser 1 5 10 15 Gln Glu Asn Asn Arg Glu His Arg Thr Glu Glu Ile Asp Arg Lys 20 25 30 Arg Leu Ile Val Arg Arg Gly Gln Ala Phe Ser Leu Thr Val His 35 40 45 Leu Ser Asp Pro Leu Gln Ser Gly His Glu Leu Ala Leu Val Leu 50 55 60 Lys Gln Asp Lys Ile Asn Asp Asp Ile Val Ile Arg Gln Arg Thr 65 70 75 Thr Gly Gly Ser Gly Asp Lys Trp Trp Leu His Gln Gln Ser Ala 80 85 90 Asn Asn Glu Leu Leu Leu Thr Val Tyr Ser Pro Ala Arg Ala Ala 95 100 105 Val Gly Glu Tyr Arg Leu Ala Val Glu Leu Met Ser Gly Asn Lys 110 115 120 Leu Leu Glu Ar g Thr Asp Phe Thr Lys Met Tyr Leu Leu Phe Asn 125 130 135 Pro Trp Cys Lys Glu Asp Ala Val Tyr Leu Pro Asp Glu Cys Leu 140 145 150 Leu Lys Glu Tyr Ile Met Asn Glu Asn Gly Arg Ile Phe Thr Gly 155 160 165 Ser Ala Asp Trp Met Ser Gly Leu Pro Trp Asn Phe Gly Gln Phe 170 175 180 Glu Asp Asn Val Met Asp Ile Cys Phe Glu Ile Leu Asp Arg Phe 185 190 195 Asn Pro Ala Arg Ser Asp Pro Pro Ser Asp Met Leu Gln Arg Trp 200 205 210 Asp Pro Val Tyr Ile Ser Arg Ala Val Val Ala Met Val Asn Ala 215 220 225 Asn Asp Asp Asp Gly Gly Val Val Val Gly Arg Trp Gln Glu Pro 230 235 240 Tyr Thr Gly Gly Val Gln Pro Thr Lys Trp Met Ser Ser Val Pro 245 250 255 Ile Leu Glu Glu Trp Ser Lys Ser Lys Ser Gly Val Lys Tyr Gly 260 265 270 Gln Cys Trp Val Phe Ala Ala Val Ala Cys Thr Val Met Arg Cys 275 280 285 Leu Gly Ile Pro Thr Arg Cys Ile Thr Asn Phe Gln Ser Ala His 290 295 300 Asp Thr Asp Gly Asn Leu Ser Ile Asp Arg Val Tyr Asn Ile His 305 310 315 Arg Gln Leu Val Asp Gly Asp Asp Ser Ile Trp Asn Phe His Cys 320 325 330 Trp Il e Glu Ser Tyr Met Gln Arg Glu Asp Leu Pro Glu Gly Tyr 335 340 345 Gly Gly Trp Gln Val Leu Asp Pro Thr Pro Gln Glu Arg Ser Ser 350 355 360 Gly Met Phe Arg Cys Gly Pro Cys Pro Leu Lys Ala Ile Lys Glu 365 370 375 Gly Asp Leu Asn Val Lys Phe Asp Val Pro Phe Val Phe Ala Glu 380 385 390 Val Asn Ala Asp Ile Ile Asn Trp Glu Ile Arg Pro Asp Gly Gln 395 400 405 Arg Lys Arg Leu Ser Ser Asn Ser Ala Asn Val Gly Arg Asn Ile 410 415 420 Ser Thr Lys Ser Pro Tyr Gly Asn Glu Arg Glu Asp Ile Thr His 425 430 435 Gln Tyr Lys Tyr Gln Glu Gly Ser Ala Lys Glu Arg Glu Val Tyr 440 445 450 Asn Lys Ala Gly Arg Arg Ile Ser Gly Pro Asp Gly Glu Glu Glu 455 460 465 Ser Lys Pro Gly Asn Val Gln Leu Glu Ile Lys His Ala Lys Pro 470 475 480 Val Phe Gly Thr Asp Phe Asp Val Ile Phe Glu Leu Glu Asn Met 485 490 495 Gly Asp Lys Glu Val Ser Cys Lys Leu Asn Met Met Ser Glu Ala 500 505 510 Val Thr Tyr Asn Ser Val His Leu Gly Arg Phe Gln Asn Ser Thr 515 520 525 Val Asn Val Val Ile Pro Ala His Lys Val His Ser Glu Thr Val 530 535 54 0 Arg Leu Leu Tyr Thr Lys Tyr Ala Ser Val Val Ser Glu His Asn 545 550 555 Ile Ile Arg Val Thr Gly Val Ala Glu Val Ser Gly Gln Glu Lys 560 565 570 Ser Ile Leu Glu Met Val Asn Ile Pro Leu Ser Lys Pro Lys Leu 575 580 585 Ser Ile Lys Val Pro Gly Trp Val Ile Leu Asn Arg Lys Ile Thr 590 595 600 Thr Phe Ile Ser Phe Thr Asn Pro Leu Pro Val Pro Leu Asn Arg 605 610 615 Gly Val Phe Thr Val Glu Gly Ala Gly Leu Leu Pro Thr Lys Glu 620 625 630 Ile Arg Ile Ser Gly Ser Ile Ala Pro Gly Gln 635 640 SEQ ID NO: 51 Sequence Length: 1921 Sequence Type: Nucleic Acid Strand Count: Double Strand Topology: Linear Sequence Type: cDNA to mRNA Origin organism name: Alaska pollack Character that characterizes muscle sequence: CDS Location: 1 .. Method of determining the feature: P sequence GCCCACACAA ACCGTTTAAT TGCTGGTGTT GATCTGAGAA GCCAGGAAAA CAACCGGGACT AG TGGAGTCACT CTTAGGAGTCATGCTAGCTGATGCTGACTGATGCTAG ACCTCTCCGA CCCGCTGCAG TCCGGCCATG AGCTGGCCCT GGTCTTAAAG 180 CAGGATAAAA TCAACGAT GA TATTGTGATC AGACAGCGAA CGACTGGAGG GTCCGGTGAC 240 AAGTGGTGGT TACACCAGCA GAGCGCGAAC AACGAATTAC TGCTGACTGT GTACAGTCCC 300 GCCCGTGCTG CCGTTGGCGA GTACCGCTTG GCTGTTGAAC TGATGTCAGG GAATAAACTT 360 CTGGAGAGGA CGGACTTTAC CAAAATGTAC TTGCTGTTTA ATCCCTGGTG CAAAGAAGAT 420 GCCGTGTACC TCCCTGATGA GTGTCTGCTC AAGGAATACA TTATGAACGA GAATGGTCGC 480 ATTTTCACTG GGAGTGCGGA TTGGATGAGT GGGTTGCCAT GGAATTTCGG ACAGTTTGAA 540 GATAATGTGA TGGACATCTG CTTTGAGATC CTTGACCGCT TTAACCCAGC GAGGTCAGAC 600 CCCCCAAGCG ACATGCTTCA GCGATGGGAC CCTGTCTACA TCAGCAGGGC AGTCGTTGCC 660 ATGGTGAATG CCAACGATGA TGACGGTGGA GTCGTGGTGG GTCGATGGCA GGAACCTTAC 720 ACAGGTGGAG TACAGCCAAC CAAATGGATG AGCAGTGTGC CCATCCTGGA AGAGTGGAGC 780 AAATCAAAGT CTGGAGTGAA ATATGGCCAA TGCTGGGTGT TTGCAGCCGT GGCCTGCACA 840 GTGATGCGAT GCCTGGGCAT CCCCACACGC TGCATCACCA ACTTTCAGTC GGCCCATGAC 900 ACAGACGGAA ACCTCTCCAT CGACCGAGTG TACAACATAC ATAGGCAGCT AGTTGACGGT 960 GATGACAGTA TCTGGAACTT TCATTGTTGG ATCGAGTCTT ACATGCAGAG AGAAGATCTA 1020 CCTGAAGGAT ATGGTGGCTG GCAAGTCTTG GACCC CACAC CTCAGGAGAG GAGTAGTGGT 1080 ATGTTTCGCT GTGGCCCATG TCCTTTGAAG GCCATTAAAG AAGGGGACCT CAATGTGAAG 1140 TTTGATGTTC CATTTGTCTT TGCTGAGGTG AATGCAGACA TCATCAATTG GGAAATCAGA 1200 CCAGACGGTC AGCGAAAGCG GCTTTCATCC AACTCTGCAA ATGTGGGGAG GAACATTAGC 1260 ACCAAAAGTC CTTATGGTAA CGAGAGGGAA GATATAACCC ATCAGTACAA GTACCAAGAA 1320 GGTTCAGCCA AGGAGCGGGA GGTGTACAAC AAGGCAGGGC GGCGCATCTC CGGGCCGGAT 1380 GGAGAAGAGG AATCAAAACC AGGAAACGTG CAGCTGGAGA TCAAGCACGC CAAACCTGTG 1440 TTCGGGACCG ACTTTGACGT CATCTTTGAG TTGGAGAACA TGGGAGACAA AGAAGTCAGC 1500 TGCAAATTAA ACATGATGTC AGAGGCTGTC ACCTATAACT CAGTTCACCT TGGACGGTTC 1560 CAGAACAGCA CGGTCAATGT TGTCATTCCT GCTCACAAAG TCCACAGTGA GACGGTGCGT 1620 CTACTCTACA CTAAGTATGC CTCAGTTGTC AGCGAGCACA ACATCATCCG GGTGACAGGG 1680 GTGGCGGAAG TGTCCGGCCA GGAAAAATCC ATCCTGGAGA TGGTCAACAT CCCACTGAGC 1740 AAGCCCAAAC TCAGTATTAA GGTTCCTGGC TGGGTGATTT TAAATAGGAA AATCACCACC 1800 TTCATCTCCT TCACCAATCC ATTGCCAGTG CCACTGAACC GAGGAGTGTT CACTGTTGAA 1860 GGGGCTGGCC TACTTCCCAC CAAAGAGATC CGCATTTCTG GTAGCATCGC TCCAGGCCAG 1920 C 1921 SEQ ID NO: 52 Sequence length: 1962 Sequence type: Nucleic acid strand number: Double-stranded topology: Linear array type: cDNA to mRNA Origin organism name: Alaska pollack muscle sequence characteristic features Symbols: CDS Location: 1 .. Method of determining features: P sequence TTGTAAGAGC AACTCTTGGA AAGAATTTAG CAAAGATA ATG GCC CAC ACA 50 Met Ala His Thr 1 AAC CGT TTA ATT GCT GGT GTT GAT CTG AGA AGC CAG GAA AAC AAC 95 Asn Arg Leu Ile Ala Gly Val Asp Leu Arg Ser Gln Glu Asn Asn 5 10 15 CGG GAA CAC CGA ACT GAG GAG ATT GAT AGG AAG CGT TTG ATT GTT 140 Arg Glu His Arg Thr Glu Glu Ile Asp Arg Lys Arg Leu Ile Val 20 25 30 CGG CGG GGA CAA GCC TTC TCC CTG ACG GTG CAC CTC TCC GAC CCG 185 Arg Arg Gly Gln Ala Phe Ser Leu Thr Val His Leu Ser Asp Pro 35 40 45 CTG CAG TCC GGC CAT GAG CTG GCC CTG GTC TTA AAG CAG GAT AAA 230 Leu Gln Ser Gly His Glu Leu Ala Leu Val Leu Lys Gln Asp Lys 50 55 60 ATC AAC GAT GAT ATT GTG ATC AGA CAG CGA ACG ACT GGA GGG TCC 275 Ile Asn Asp Asp Ile Val Ile Arg Gln Arg Thr Thr Gly Gly Ser 65 70 75 GGT GAC AAG TGG TGG TTA CAC CAG CAG AGC GCG AAC AAC GAA TTA 320 Gly Asp Lys Trp Trp Leu His Gln Gln Ser Ala Asn Asn Glu Leu 80 85 90 CTG CTG ACT GTG TAC AGT CCC GCC CGT GCT GCC GTT GGC GAG TAC 365 Leu Leu Thru Val Tyr Ser Pro Ala Arg Ala Ala Val Gly Glu Tyr 95 100 105 CGC TTG GCT GTT GAA CTG ATG TCA GGG AAT AAA CTT CTG GAG AGG 410 Arg Leu Ala Val Glu Leu Met Ser Gly Asn Lys Leu Leu Glu Arg 110 115 120 ACG GAC TTT ACC AAA ATG TAC TTG CTG TTT AAT CCC TGG TGC AAA 455 Thr Asp Phe Thr Lys Met Tyr Leu Leu Phe Asn Pro Trp Cys Lys 125 130 135 GAA GAT GCC GTG TAC CTC CCT GAT GAG TGT CTG CTC AAG GAA TAC 500 Glu Asp Ala Val Tyr Leu Pro Asp Glu Cys Leu Leu Lys Glu Tyr 140 145 150 ATT ATG AAC GAG AAT GGT CGC ATT TTC ACT GGG AGT GCG GAT TGG 545 Ile Met Asn Glu Asn Gly Arg Ile Phe Thr Gly Ser Ala Asp Trp 155 160 165 ATG AGT GGG TTG CCA TGG AAT TTC GGA CAG TTT GAA GAT AAT GTG 590 Met Ser Gly Leu Pro Trp Asn Phe Gly Gln Phe Glu Asp Asn Val 17 0 175 180 ATG GAC ATC TGC TTT GAG ATC CTT GAC CGC TTT AAC CCA GCG AGG 635 Met Asp Ile Cys Phe Glu Ile Leu Asp Arg Phe Asn Pro Ala Arg 185 190 195 TCA GAC CCC CCA AGC GAC ATG CTT CAG CGA TGG GAC CCT GTC TAC 680 Ser Asp Pro Pro Ser Asp Met Leu Gln Arg Trp Asp Pro Val Tyr 200 205 210 ATC AGC AGG GCA GTC GTT GCC ATG GTG AAT GCC AAC GAT GAT GAC 725 Ile Ser Arg Ala Val Val Ala Met Val Asn Ala Asn Asp Asp Asp 215 220 225 GGT GGA GTC GTG GTG GGT CGA TGG CAG GAA CCT TAC ACA GGT GGA 770 Gly Gly Val Val Val Gly Arg Trp Gln Glu Pro Tyr Thr Gly Gly 230 235 240 GTA CAG CCA ACC AAA TGG ATG AGC AGT GTG CCC ATC CTG GAA GAG 815 Val Gln Pro Thr Lys Trp Met Ser Ser Val Pro Ile Leu Glu Glu 245 250 255 TGG AGC AAA TCA AAG TCT GGA GTG AAA TAT GGC CAA TGC TGG GTG 860 Trp Ser Lys Ser Lys Ser Gly Val Lys Tyr Gly Gln Cys Trp Val 260 265 270 TTT GCA GCC GTG GCC TGC ACA GTG ATG CGA TGC CTG GGC ATC CCC 905 Phe Ala Ala Val Ala Cys Thr Val Met Arg Cys Leu Gly Ile Pro 275 280 285 ACA CGC TGC ATC ACC AAC TTT CAG TCG GC C CAT GAC ACA GAC GGA 950 Thr Arg Cys Ile Thr Asn Phe Gln Ser Ala His Asp Thr Asp Gly 290 295 300 AAC CTC TCC ATC GAC CGA GTG TAC AAC ATA CAT AGG CAG CTA GTT 995 Asn Leu Ser Ile Asp Arg Val Tyr Asn Ile His Arg Gln Leu Val 305 310 315 GAC GGT GAT GAC AGT ATC TGG AAC TTT CAT TGT TGG ATC GAG TCT 1040 Asp Gly Asp Asp Ser Ile Trp Asn Phe His Cys Trp Ile Glu Ser 320 325 330 TAC ATG CAG AGA GAA GAT CTA CCT GAA GGA TAT GGT GGC TGG CAA 1085 Tyr Met Gln Arg Glu Asp Leu Pro Glu Gly Tyr Gly Gly Trp Gln 335 340 345 GTC TTG GAC CCC ACA CCT CAG GAG AGG AGT AGT GGT ATG TTT CGC 1130 Val Leu Asp Pro Thr Pro Gln Glu Arg Ser Ser Gly Met Phe Arg 350 355 360 TGT GGC CCA TGT CCT TTG AAG GCC ATT AAA GAA GGG GAC CTC AAT 1175 Cys Gly Pro Cys Pro Leu Lys Ala Ile Lys Glu Gly Asp Leu Asn 365 370 375 GTG AAG TTT GAT GTT CCA TTT GTC TTT GCT GAG GTG AAT GCA GAC 1220 Val Lys Phe Asp Val Pro Phe Val Phe Ala Glu Val Asn Ala Asp 380 385 390 ATC ATC AAT TGG GAA ATC AGA CCA GAC GGT CAG CGA AAG CGG CTT 1265 Ile Ile Asn Trp Glu Ile Arg Pro Asp Gly Gln Arg Lys Arg Leu 395 400 405 TCA TCC AAC TCT GCA AAT GTG GGG AGG AAC ATT AGC ACC AAA AGT 1310 Ser Ser Asn Ser Ala Asn Val Gly Arg Asn Ile Ser Thr Lys Ser 410 415 420 CCT TAT GGT AAC GAG AGG GAA GAT ATA ACC CAT CAG TAC AAG TAC 1355 Pro Tyr Gly Asn Glu Arg Glu Asp Ile Thr His Gln Tyr Lys Tyr 425 430 435 CAA GAA GGT TCA GCC AAG GAG CGG GAG GTG TAC AAC AAG GCA GGG 1400 Gln Glu Gly Ser Ala Lys Glu Arg Glu Val Tyr Asn Lys Ala Gly 440 445 450 CGG CGC ATC TCC GGG CCG GAT GGA GAA GAG GAA TCA AAA CCA GGA 1445 Arg Arg Ile Ser Gly Pro Asp Gly Glu Glu Glu Ser Lys Pro Gly 455 460 465 AAC GTG CAG CTG GAG ATC AAG CAC GCC AAA CCT GTG TTC GGG ACC 1490 Asn Val Gln Leu Glu Ile Lys His Ala Lys Pro Val Phe Gly Thr 470 475 480 GAC TTT GAC GTC ATC TTT GAG TTG GAG AAC ATG GGA GAC AAA GAA 1535 Asp Phe Asp Val Ile Phe Glu Leu Glu Asn Met Gly Asp Lys Glu 485 490 495 GTC AGC TGC AAA TTA AAC ATG ATG TCA GAG GCT GTC ACC TAT AAC 1580 Val Ser Cys Lys Leu Asn Met Met Ser Glu Ala Val Thr Tyr A sn 500 505 510 TCA GTT CAC CTT GGA CGG TTC CAG AAC AGC ACG GTC AAT GTT GTC 1625 Ser Val His Leu Gly Arg Phe Gln Asn Ser Thr Val Asn Val Val 515 520 525 ATT CCT GCT CAC AAA GTC CAC AGT GAG ACG GTG CGT CTA CTC TAC 1670 Ile Pro Ala His Lys Val His Ser Glu Thr Val Arg Leu Leu Tyr 530 535 540 ACT AAG TAT GCC TCA GTT GTC AGC GAG CAC AAC ATC ATC CGG GTG 1715 Thr Lys Tyr Ala Ser Val Val Ser Glu His Asn Ile Ile Arg Val 545 550 555 ACA GGG GTG GCG GAA GTG TCC GGC CAG GAA AAA TCC ATC CTG GAG 1760 Thr Gly Val Ala Glu Val Ser Gly Gln Glu Lys Ser Ile Leu Glu 560 565 570 ATG GTC AAC ATC CCA CTG AGC AAG CCC AAA CTC AGT ATT AAG GTT 1805 Met Val Asn Ile Pro Leu Ser Lys Pro Lys Leu Ser Ile Lys Val 575 580 585 CCT GGC TGG GTG ATT TTA AAT AGG AAA ATC ACC ACC TTC ATC TCC 1850 Pro Gly Trp Val Ile Leu Asn Arg Lys Ile Thr Thr Phe Ile Ser 590 595 600 TTC ACC AAT CCA TTG CCA GTG CCA CTG AAC CGA GGA GTG TTC ACT 1895 Phe Thr Asn Pro Leu Pro Val Pro Leu Asn Arg Gly Val Phe Thr 605 610 615 GTT GAA GGG GCT GGC CTA CT T CCC ACC AAA GAG ATC CGC ATT TCT 1940 Val Glu Gly Ala Gly Leu Leu Pro Thr Lys Glu Ile Arg Ile Ser 620 625 630 GGT AGC ATC GCT CCA GGC CAG C 1962 Gly Ser Ile Ala Pro Gly Gln 635 640 SEQ ID NO: 53 Sequence length: 4 Sequence type: Amino acid topology: Linear Sequence type: Peptide fragment type: Intermediate fragment Origin organism name: Fish SEQ ID NO: 54 sequence length: 6 sequence type: amino acid topology: linear sequence type: peptide fragment type: intermediate type fragment origin organism name: fish SEQ ID NO: 55 sequence length: 6 sequence type: amino acid topology: linear sequence type: peptide fragment type: intermediate fragment origin organism name: fish SEQ ID NO: 56 sequence length: 7 sequence type: amino acid topology: linear sequence type: peptide fragment type: intermediate fragment origin organism name: fish SEQ ID NO: 57 sequence length: 12 sequence type: amino acid topology: linear sequence type: peptide fragment type: intermediate fragment origin of organism: fish SEQ ID NO: 58 Sequence Length: 7 Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Fragment Type: Intermediate Fragment Origin Organism Name: Fish SEQ ID NO: 59 sequence length: 10 sequence type: amino acid topology: linear sequence type: peptide fragment type: intermediate fragment origin of organism: fish SEQ ID NO: 60 sequence length: 10 sequence type: amino acid topology: linear sequence type: peptide fragment type: intermediate fragment origin of organism: fish SEQ ID NO: 61 sequence length: 6 sequence type: amino acid topology: linear sequence type: peptide fragment type: intermediate fragment origin of organism: fish SEQ ID NO: 62 sequence length: 6 sequence type: amino acid topology: linear sequence type: peptide fragment type: intermediate fragment origin organism name: fish SEQ ID NO: 63 sequence length: 4 sequence type: amino acid topology: linear sequence type: peptide fragment type: intermediate fragment origin of organism: fish SEQ ID NO: 64 sequence length: 9 sequence type: amino acid topology: linear sequence type: peptide fragment type: intermediate fragment origin organism name: fish SEQ ID NO: 65 sequence length: 30 sequence type: nucleic acid chain number: single-stranded topology: linear sequence type: other nucleic acid synthetic DNA sequence TTGGAAGCTT GTAAGAGCAA CTCTTGGAAA 30 SEQ ID NO: 66 sequence length: 25 Sequence type: Nucleic acid strand number: Single-stranded topology: Linear sequence type: Other nucleic acid Synthetic DNA sequence TTGTACACTC GATCGATGGA GAGGT 25 SEQ ID NO: 67 Sequence length: 24 Sequence type: Number of nucleic acid strands : Single-stranded topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence TCTGCTTTGG GATCCTTGAC CGCT 24 SEQ ID NO: 68 Sequence length: 23 Sequence type: Nucleic acid chain number: Single-stranded topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence TGAAGGAGAG CTCCACAGAC ACA 23 SEQ ID NO: 69 Sequence length: 20 Sequence type: Nucleic acid Strand number: Single-stranded topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence ATGATGTCAA AGGCTGTCAC 20 SEQ ID NO: 70 Sequence length: 20 Sequence type: Number of nucleic acid strands: Single-stranded topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence TCTTACCATA TAAGTTGTAA 20 SEQ ID NO: 71 Sequence length: 20 Sequence type: Number of nucleic acid strands: One Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence ATTGATTAAC AACAAAATGG 20

[Brief description of drawings]

FIG. 1 Restriction enzyme map of the obtained plasmid pSLTG5 carrying the cDNA having the gene encoding transglutaminase of red sea bream

[Fig. 2] Correlation between cDNA clones having a gene encoding transglutaminase of Alaska pollack and restriction enzyme map of cDNA.

FIG. 3: Construction process diagram and restriction enzyme diagram of expression plasmid pIL6TG1 of red sea bream transglutaminase cDNA

FIG. 4 shows the nucleotide sequence of chemically synthesized DNA-1 showing the consensus SD (Shine-Dalgarno) sequence of Escherichia coli and the DNA nucleotide sequence encoding methionine at the amino terminal of red sea bream transglutaminase to leucine at the 32nd position. .

FIG. 5: Construction process diagram of plasmid pFTGN6 for construction of expression plasmid pTTG2-22 of red sea bream transglutaminase cDNA

FIG. 6 is a process chart of obtaining DNA fragments A, B, and C for constructing expression plasmid pTTG2-22 of red sea bream transglutaminase cDNA.

FIG. 7: Construction process diagram of plasmid pTTG1

FIG. 8: Construction process diagram of expression plasmid pTTG2-22 of red sea bream transglutaminase cDNA

FIG. 9: Construction process diagram of expression plasmid pYSTG1 in yeast of red sea bream transglutaminase cDNA

FIG. 10: Restriction map of transglutaminase cDNA clone derived from Alaska pollack muscle

FIG. 11: Restriction enzyme map of cDNA encoding flounder transglutaminase

FIG. 12: Plasmid having cDNA fragment encoding flounder transglutaminase

FIG. 13: Temperature stability comparison between Alaska pollack-derived transglutaminase (FTG) and guinea pig transglutaminase (MTG) ○-○, relative residual activity of FTG ●-●, relative residual activity of MTG

FIG. 14: Reactivity comparison of Alaska pollack-derived transglutaminase (FTG) and guinea pig-derived transglutaminase (MTG) in polymerization reaction of myosin H chain Δ- △, FTG ○-○, MTG ●-●, no addition control.

Front page continuation (51) Int.Cl. ⁷ Identification code FI C12N 9/10 C12R 1:19 C12P 21/02 C12N 15/00 ZNAA // (C12N 9/10 5/00 A C12R 1:19) (72 ) Yutaka Matsui 1-1, Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Kanagawa Ajinomoto Co., Inc. Central Research Laboratory (56) References Chemical Abstracts, Vol. 113, No. 21 (1990), abstract no. 189898q (58) Fields surveyed (Int.Cl. ⁷ , DB name) C12N 15/00-15/54 C12N 9/10 GenBank / EMBL / DDBJ / GeneSeq SwissProt / PIR / GeneS eq

Claims (10)

(57) [Claims] 1. A DNA fragment having a gene encoding a polypeptide having transglutaminase activity derived from fish, which comprises the amino acid sequence of 694 residues shown in SEQ ID NO: 2 in the Sequence Listing. 2. The DNA fragment according to claim 1, which has the base sequence shown in SEQ ID NO: 4 in the Sequence Listing. 3. A DNA fragment having a gene encoding a polypeptide having a transglutaminase activity derived from fish, which comprises the amino acid sequence of 695 residues shown in SEQ ID NO: 6 of the Sequence Listing. 4. The DNA fragment according to claim 3, which has the base sequence shown in SEQ ID NO: 8 in the Sequence Listing. 5. A DNA fragment having a gene encoding a polypeptide having a transglutaminase activity derived from fish, which comprises the amino acid sequence of 687 residues shown in SEQ ID NO: 43 of the Sequence Listing. 6. The DNA fragment according to claim 5, which has the nucleotide sequence shown in SEQ ID NO: 45 of the Sequence Listing. 7. A recombinant plasmid incorporating the DNA fragment according to any one of claims 1 to 6. 8. The recombinant plasmid according to claim 7, wherein the plasmid is an expression vector. 9. A transformant transformed with the recombinant plasmid according to claim 7. 10. A method for producing a polypeptide having a transglutaminase activity derived from fish, which comprises culturing the transformant according to claim 9.