JPS6091980A - Production of microorganism for developing protease - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention describes the use of plasmids that contain DNA sequences that direct the expression and secretion of specific classes of proteases and that readily convert microorganisms to overproduce and secrete specific proteases or other proteins. structure.

Concerning isolation and confirmation.

Proteases are a group of enzymes that hydrolyze peptide bonds in proteins. Proteases produced by bacteria can be broken down into two general types.

Those that are active at neutral pH and require a cofactor, such as zinc, to be active are called neutral proteases, and ethylenediaminetetraacetic acid (ethylenediaminetetraacetic acid), which removes the cofactor from the enzyme, is called a neutral protease.
Inactivated by chelating agents such as EDTA). Those that are active at high pH and cleave peptide bonds by a process similar to alkaline hydrolysis are called alkaline glothiases. Alkaline proteases are also interpreted as subtiliain and serine proteases. Alkaline glothiase is phenylmethylsulfonyl fluoride (PMSF) or diimpropylfluorophosphinyl fluoride (DF
P) is inactivated by chemicals such as P). Bacterial proteases are available from numerous companies (OPD Chemical
Buyers Directory, A, P, Kava
ler, ed, Schnell Pcblishing
Co.

Inc., New York 1982) and is used to clarify beer, tan skins, tenderize meat, and
Used to curdle milk and as a component of certain detergents. Bacterial enzymes are extracted from culture media of specific organisms, many of which are the result of careful selection and artificial mutagenesis (eg, UV irradiation).

Limits on enzyme production include the rate at which the microorganism produces the enzyme, the extent to which the microorganism excretes the enzyme produced, and the stability of the microorganism in the culture medium.

Recently, processes have been developed in which genes from another microorganism are inserted into bacterial organisms so that the bacteria produce "foreign" proteins. The technique of introducing DNA that directs the production of enzymes (``specifying genetic information (codos for)'') involves attaching (``ligating'') split DNA to DNA from a plasmid to create a "vector."
ting)'') and introducing the vector into the host under conditions that allow for successful "conversion".

Microorganisms of the genus Bacillus are aerobic bacteria and are nonpathogenic, so they are widely used in the fermentation industry and for protein production because they have the ability to secrete proteins. genetic hybrid DN
Many studies of A. coli (E., colt.
) has been used. Escherichia coli (E. coli) is a non-secreting microorganism that is generally well-developed for cloning genetic hybrid plasmids, but is not well suited for commercial production of proteins. Bacillus species, particularly B. subtilis, cannot be transformed using monomeric plasmids or conventional techniques developed for E. coli.
) etc., Mol. gen. Genet. , 166:259
[1978], Kaggins et al., Proc. Nat.
Acad. SeiU. S. , 75:1423 [1978
]). and Michel et al. , Gone, 12:14
7 [1978]). Use of plasmid multimers (Cano
si), the use of polyethylene glycol to induce DNA uptake into protoplasts (Chang S. and Coh
enS. N.

Mol. gen. Genet, 168:111 [19
[79]) and the use of marker-rescue methods (Gry
Czan et al., Mol. gen. Gonet.

A number of alternative strategies have been developed for transformation of B. subtilis, including B. subtilis (B. 177:459 [1980]).

B, amyloliquefaci containing relevant control regions
The structural gene for α-amylase from E. ens was shotgun cloned into B. subtilis. α-Amylase is urinary bacteria B, am
was expressed in B. subtilis (Polva I) at a rate five times faster than that produced by B. ylolique faaiena.

Gene, 19:81-87 [1982]).

Bacillus transformants, such as Bacillus subtilis (B.
) transformants express large amounts of propase and may be of particular commercial significance. In addition, B. subtilis (
An effective expression vector for transforming B. subtilis is Bacillus subtilis (B. subtilis) to produce the enzyme.
Not only because it can be used to transform B. subtilis (B. It would be useful.

The present invention provides that the genetic information useful for regulating the expression of the protease of the first Bacillus species, such as B. amyloliquefaciens, is derived from the protease or, if desired, other heterologous polypeptides, i.e., not naturally produced by the Bacillus host. Bacillus subtilis (B, sub
tilis). Accordingly, the present invention provides for the production of (a) a replicon; (b) a promoter and regulatory region that regulates the expression and secretion of the rus protease. and (C) a DNA sequence encoding the amino acid sequence of a polypeptide linked to the promoter and regulatory region.
Replicable grasmid expression vectors have been provided that are capable of directing high level expression and secretion of polypeptides in transformed Bacilli, such as Bacillus subtilis.

In one embodiment of the invention, the encoded polypeptide is linked to a particular promoter and regulatory region, such as B, amyloliq.
uefaciens is a protease controlled by the promoter and regulatory region for the alkaline glothiase or neutral protease gene.

In another embodiment, the encoded polypeptide is a heterologous polypeptide that is not normally under the control of a protease promoter and regulatory region. The heterologous polypeptide may be considered a prokaryotic protein, eg protein A1, or a eukaryotic protein, eg prorennin.

In accordance with the present invention there is also provided a transformant microorganism comprising a replicable plasmid expression vector as described above; 1) a method for producing a polypeptide by expression of NA for encoding a polypeptide in a transformant microorganism; and the present invention The polypeptide encoded by the replicable plasmid expression vector-1 DNA sequence of B, amyloliquef
aciens protease is preferred.
A method of producing a microorganism expressing a protease enzyme comprising transforming a bacillus, such as B. subtilis, by a method of producing a microorganism expressing a protease enzyme.

The replicable plasmid expression vector of the present invention is a DNA
It is made by inserting the DNA sequences described below into a plasmid that can be transformed and replicated in a host microorganism using recombinant methods.

(Unless otherwise stated, all DNA referred to herein is in the form of double-stranded DNA.) Plasmids are non-chromosomal forms of double-stranded DNA that are often found in microorganisms in multiple copies per cell. It is a sex loop.

A plasmid contains within its DNA sequence the genetic information (i.e., a "replicon") necessary to replicate itself. Additionally, many plasmids are useful if one wishes to screen for the presence of a particular plasmid. , contains sequences of DNA that encode phenotypic purpura, such as resistance to certain antibiotics.

Plasmids that are capable of transforming and replicating themselves in a host microorganism can be used in the practice of this invention. When the host microorganism is Bacillus subtilis (B, sub-T), plasmid pBD64 is used to create a replicable plasmid expression vector, and B. B,
subtilis). Both plasmids are described by Gryczan et al., supra. A homologous plasmid is an endogenous plasmid in B. subtilis that has large regions of DNA sequence homology with the exogenous plasmid that it is desired to introduce and that can be recombined by the exogenous plasmid during transformation. The endogenous plasmid pUB110 is pBD6
It is a homologous plasmid to 4.

The use of endogenous homologous plasmids to increase the efficiency of transformation was used in carrying out the invention by Grycz.
This method is described in a paper by An et al., and is known as the marker rescue conversion method.

The DNA sequence inserted into the plasmid to generate the replicable plasmid expression vector of the invention is:
B, amyloli-quef bound to the NA sequence
It contains a DNA sequence consisting of a promoter and a linkage region that controls the expression and secretion of protease in P. aciens. In one embodiment, the encoded polypeptide is B, amyloliquefaciens protease (alkaline or neutral).

A promoter is a sequence of deoxyribonucleotides that is identified and linked by the enzyme RNA polymerase and may be overlapped by a so-called operator sequence. The operator sequence is recognized and bound by a repressor protein that is absent or inactive under conditions of expression. RNA polymerase binds to the promoter and then moves along the coding strand of DNA (unless blocked by a bound repressor protein) copying the base sequence into the corresponding mRNA. The part of the DNA that is copied is Shl
The liposome binding site known as the ne-Dalgarno sequence is symbolized. This is followed by a translation initiation signal, usually ATG, in the DNA sequence, which is a nucleotide triplet symbolizing the first amino acid in the structural gene. The remainder of the transcript encodes the polypeptide gene. RNA polymerase action is caused by DNA
It ends when the stop signal in A is encountered. The resulting mRNA is efficiently translated in liposomes to create the desired protein. In the case of secreted proteins such as α-amylase and protease, promoters,
In addition to the operator and liposome binding site, there is also a "signal sequence" that follows the translation initiation signal. this is,
A hydrophobic domain encodes a polypeptide of approximately 15-30 amino acids in length that typically includes a positively charged amino acid terminal domain followed by a bale. The signal sequence that precedes the amino acid sequence of the mature protein is required for secretion of the protein across the cell membrane and is removed from the secreted protein during or shortly after translocation across the cell membrane.

The order of the fromotor, operator, liposome binding site, and translation initiation sequence helps control the efficiency of protein expression. As used herein, the term "promoter and regulatory region" implies a DNA sequence that includes bases encoding promoters, operators, liposome binding sites, and translation initiation signals, as well as secretory signal sequences. .

The DNA sequences mainly incorporated into the replicable plasmid expression vector of the present invention (those that control the expression and secretion of the promoter and regulatory enzymes). B, am
It encodes the amino acid sequence of a protease whose expression and secretion are controlled by a specific promoter and sequence of yloliquefaions. However, DNA sequences encoding other useful heterologous polypeptides can be prepared using known methods of DNA recombination.
and inserted into a plasmid to create a replicable plasmid expression vector used for direct expression and secretion of heterologous polypeptides in B. subtilis. It will be recognized that Typical of such useful polypeptides are eukaryotic polypeptides such as human interferon, insulin, human and animal growth hormone, prorennin, etc., and prokaryotic polypeptides such as protein A. These are examples of polypeptides with known amino acid sequences expressed by genetic hybrid DNA techniques in E. coli. Bacillus subtilis (B, su
An effective means of expressing these polypeptides in E. coli has obvious advantages over expression in E. coli, such as secretion of the polypeptides from the microorganism, which simplifies purification and increases yield, and ) Eliminating the risk of contamination of products with endotoxins.

DNA sequences encoding promoters and regulatory regions and, if desired, B, amyloliquefael
The DNA sequence encoding ens protease is B, a
mylo-11quefaclena chromosa A
It can be obtained by direct division of a segment containing the desired sequence from DNA.

Alternatively, B transcribed from the desired DNA sequence.

mRNA can be isolated from C. amyloliquefaciens and used as a template to create cDNA in the presence of reverse transcriptase. Then e
The DNA is used to isolate the desired promoter, regulatory regions and structural genes.

Chromosomal DNA or cDNA can be inserted into a clonogenic vector used to transform a host microorganism. The transformant is then grown to achieve the desired D
Create a large population of clones containing the NA sequence.

Bacillus DNA, e.g. B, amyloliquef
To insert the aciens DNA into a plasmid,
The DNA must have complementary ends to those that occur at the endonuclease cleavage site of the plasmid, conveniently located for expression. This includes B, a with the restriction endonuclease corresponding to the desired insertion site of the plasmid.
mylollquefaciens by completely or partially digesting the ends of DNA segments. D symbolizing the promoter and regulatory region
The NA sequence and the amino acid sequence of the polypeptide are T4 DN
It is inserted into a plasmid (which has been linearized by cleavage with a suitable restriction endonuclease) by reaction in the presence of a ligating enzyme such as A ligase.

The resulting plasmid is used to transform a host Bacillus microorganism, which can be screened for the presence of an expression vector with the desired DNA sequence in the appropriate orientation for expression. Conversion can be carried out using known methods (Contente, S. and Dubn
au, D. Plasmid, 2:555-571 (19
79) and Chang & Cohen, supra).

Transformants can be screened for the presence of the desired genetic hybrid plasmid by assaying for the presence of an activity characteristic of the polypeptide encoded by the DNA sequence.

In the case of the DNA sequence encoding B. amyloliquefaciens protease, the presence of the desired genetic hybrid can be determined by the ability of the transformant cells to clear skim milk in agar.

If desired, replicable plasmid expression vectors can be removed from the screened transformants by known methods, such as the boil preparation method, and used to transform other host bacilli or to express polypeptides. Directly screened transformants can be used. If one wishes to express a polypeptide in E. coli, a plasmid capable of efficient replication in E. coli, e.g., plasmid pB.
R322, to split the replicable plasmid expression vector.

It may be better to use a "shuttle vector" that can be produced by recombination.

Since E. coli is an excellent host for cloning, it is desirable to prepare a shuttle vector from a replicable plasmid expression vector for the purpose of replicating it in E. coli. There is. Next, the cloned vector was used in Escherichia coli (E.
A replicable plasmid expression vector isolated from Bacillus subtilis (B.

subtilis) or other hosts.

The replicable plasmid expression vector of the present invention is Bacillus subtilis (
It can be used to transform stage zero sporulating mutants of E. coli. In fermentation, Bacillus subtilis (B, subti
The use of stage zero sporulating variants of S. lis) offers the theoretical advantage of better control of environmental contamination. Existing stage zero sporulating species produce very low levels of proteases. The plasmid produced and isolated by the above method is a Bacillus subtilis (B. subtilis).
is) is easily introduced into stage zero sporulating mutants of Bacillus subtilis (B, subtllia) using the protoplast transformation method used to transform other species. Transformed stage zero sporulating B. subtilis produced specific proteases at rates qualitatively indistinguishable from other transformed Bacillus subtilis.

The cell retains its property of not being able to produce spores.

The polypeptide encoded by the DNA sequence in a replicable plasmid expression vector is produced by growing the transformed microorganism under known fermentation conditions until the desired cell density is achieved and subjecting the transformant to conditions that initiate expression. It can be created by placing it. Addition of inexpensive carbon sources such as soy protein, fish meat, or readily available and economical denatured proteins will promote expression.

The expressed polypeptide is then recovered from other proteins and contaminants in the fermented broth and purified using known methods such as size exclusion chromatography, immunoaffinity chromatography, and the like.

In one embodiment of the invention, B. amyloli
A replicable plasmid expression vector encoding B.-quefaciens protease is created and used to transform B. subtilis in the following manner.

B, Cellular DNA from amyloliquefaction was isolated using standard methods and M
It is partially digested by a restriction endonuclease such as boI. This is then applied to the vector, namely MboI
It is ligated into the BamHI site of pBD64 with ends complementary to those left by the digestion. In the specific example invaded, B, amyloliquefac
ienn DNA was partially digested with MboI and ligated into BamHI of pBD64, which contains markers for chloramphenicol and kanamycin resistance. pB
A plasmid homologous to D64 is pUBllo. (B within the desired insert, amyloliquiofaien
If the S division site is not cut, other combinations of homologous plasmids and suitable restriction endonucleases are suitable. ) Transformed cells expressing proteases are confirmed by growing the cells on agar containing skim milk. The colonies that give rise to the zone of milk clarification are Bacillus subtilis (B, s
The desired colonies are easily selected and grown in shaker flasks. If the plasmid marker is for antibiotic resistance, antibiotics are added to the growth medium to select for colonies that do not carry the plasmid. If pBD64 is used as a plasmid, chloramphenicol is a suitable antibiotic. Clones exhibiting the selected characteristics are expanded for further testing. The plasmid containing the gene for protease expression was prepared using the boiling method (olmen).

D, S and M, Quigiey, Anal, Bioc.
hem, 114-193 [1981]). The isolated plasmid readily retransforms B. subtilis and expresses both the protease and selection marker (ie, antibiotic resistance). Alternatively, the isolated plasmid can be cloned in a host such as E. coli (E, coil) after being split and ligated into a suitable vector for E. coli transformation, such as the shuttle vector described above. generate. When such a shuttle vector is generated, the ligated halves can be oriented in either direction, allowing the clonogenic plasmid to be screened at some point to screen for non-functional configurations. preferable.

B, amyloliquofaclons contain structural genes encoding both neutral and alkaline proteases.

B. subtilis (B. aubtilks) transformed as previously described.
) will express either protease type by means of specific structural genes incorporated into a replicable plasmid expression vector. B. amyloliquaf
When vectors encoding the production of aaiens were created in this manner, two different plasmids encoding alkaline glothiase and one plasmid for neutral protease were isolated. According to restriction mapping of isolated plasmids, the difference between the two alkaline protease grasmids appears to be the inclusion of an available untranslated 600 base pair sequence at the 3' end of the structural gene encoding the protease. It will be done. No other functional or chemical differences were apparent, and the image-distorted white matter was roughly equal in activity. It should be assumed that small, non-functional differences in plasmids and proteases may be obtained when this method is used to create replicable plasmid expression vectors and transformant microorganisms.

The replicable plasmid expression vectors produced by the method described herein are B. subtilis
were compared for their ability to direct overproduction of propase. The rate of production was compared to that of the original plasmid pBD6.
4 and DNA inserts are obtained, B, amylo--l
Bacillus subtilis (B.
, aubtilis). The rate of digestion of dye-protein complexes such as Azul leather provides a convenient method of comparison. Under the same conditions, B, amy
Bacillus subtilis (B, sub
The B. tllis strain had a production rate 150-200 times higher than the B. subtilin strain transformed with pBD64; amylolique faciens
The production rate was 1.5 to 2 times higher. Neutral protease, which forms the medium, had a slightly higher production rate than alkaline protease, which forms transformants in this test.

A replicable plasmid expressing the Iaciens grotease pro polypeptide is placed under the control of the promoter and regulatory regions of the alkaline protease (apy [BamP]) or neutral protease (npr [BamP]) genes. A vector is constructed. The expression vector is Bacillus subtilis (B)
, which directs the expression and secretion of foreign proteins.

We isolated and sequenced both apr[BamP] and npr[:BamP]. B. amyloliquef
The DNA sequences and deduced amino acid sequences of alkaline protease and neutral protease from P. aciens are shown in Figures 3 and 4, respectively. In both cases, the DNA sequences showed a large open reading frame preceded by a sequence encoding the mature protease. The deduced amino acid sequence of each gene included a signal sequence and an additional polypeptide sequence (the "pro" sequence) preceding the mature protein. We are -107 for t apr (BamP), -221 for npr (BamP], the starting point of translation as an amino acid residue.
It was confirmed that in vivo apr[:1
i? that the starting point of translation of [3amP] is codon-107? l[: For clarity, codon-103 (8AA
) was changed to J, Rioker (TAA) for mutagenesis (Zoller and Smlt).
h, Nuc, Ac1dIIRes,, I(L64
87-6500 [1982]). Is alkaline glothiase host bacterial? apr[
◎ The expression vector of the invention, which is capable of directing the expression and secretion of a heterologous polypeptide in B. subtilis, is constructed from this manufacturer variant of BamP]. It can be constructed by inserting a structural gene for a heterologous protein into either ShiraP or BiCB amP3. Hybrid DNA specifying the genetic information for the signal peptide of the heterologous polypase to an appropriate plasmid is generated from Bacillus subtilis (B. subt.
ilis). A fusion protein consisting of a signal peptide and a heterologous polypeptide is produced in Bacillus subtilis (B, subttlig) by concomitant cleavage of the signal peptide to release the heterologous polypeptide into the surrounding medium.
It will be expressed and secreted from the A village that specifies the genetic information of the pro2 region of protease [Silla P1 or! ]
If any part of the sequence of the ``Pro'' region is left intact against the flow from the gene specifying the genetic information of the heterologous polypeptide, then that encoded portion of the ``pro'' region - will remain fused to the terminus.''The pro' region was previously associated only with eukaryotic proteases, so apr[BamP] and npr
The discovery of the brocoding region in [BamP] was somewhat urgent. Furthermore, the possible role of the "pro" region in promoting secretion or protecting the protein from degradation during secretion was not clear. Therefore, apr[-BamP] or npr[BamP
It was an unexpected finding that secretion of the expression product of a heterologous gene occurs in B. subtilis without direct binding to the signal-coding sequence of the f1 or 'pro' region. A putative signal processing site between the blur and pro regions was identified by Perlman and H.
alvorson (J.

Mol. Biasl. 167:391-4091 [19
[83]), it was confirmed on the basis of a cooperating 'signal sequence'. Signal processing sites can be determined by sequencing the amine termini of secreted proteins. Once it is determined whether the signal processing is against or along the flow of the issued signal, the heterogeneous fusion is modified accordingly.

Insertion of a structural gene into a heterologous polypeptide along the line from the signal peptide coding region involves inserting an endonuclease cleavage site exactly along the line from the signal peptide coding region that is compatible with the cleavage site on the gene for the desired heterologous polypeptide. To create apr[:BamP] ;J>rui or npr[BamP
] Oligonucleotide-designated sequence 5103-5112 [
1983]). For example, we write the fifth block of the block coding region as follows:
A BamHI cleavage site flush with the signal coding sequence was created by inserting 6 deoxyribonucleotides after the deoxyribonucleotide of:
Mutated Sequence The mutated apr[:BamP] gene is split at the BamHI site and contains the alkaline protease pro region (GCA GG) followed by the protein A sequence starting at amino acid 23.
protein A from Staphylococcus aureus (S, aursus) to create a fusion gene specifying the genetic information of the signal peptide and first two amino acids of the protein A (encoded by G).
A compatible BclI site was linked to the 22 codon structural gene. A plasmid containing this fusion gene was used to transform B. subtilis. Transformants containing the plasmid were propagated and demonstrated to correspond immunologically to protein A by enzyme-linked immunoabsorbent assay and immunoplot analysis.

Genes for other heterologous polypeptides are apr[Bam
P] or apr[BamP] and inserted into B. subtilis (
B, subtills).

The invention is further illustrated by the following examples: Example I Construction of a replicable plasmid expression vector containing a structural gene for a protease and the construction of a B. subtilis
is) conversion of Bacillus amylolique
faciens (ATCC23844) is Saito,
H and Miura (Biochim. Biophys. A
cta, 72:619 [1963]) and partially digested with Mbol (Old, R.W. and Primrose, S.B.).

“Principles of genetic dry cropping”, 2nd ed, Univ.
of Colif, Presg., Berkeley, 19
81). B. 250 μg of amyloliquefaciens chromosomal DNA was treated with 192 units of MboI at 37°C.
Digested for 7 minutes. Digestion was terminated by phenol:chloroform extraction and DNA was precipitated with ethanol. For ligation, use 2μg of DNA/10ug, T4
Add 1 unit of ligase to chromosomal DNA at a 1:1 ratio.
: performed by using pBD64 plasmid and incubating for 60 minutes at room temperature (~25°C), -7
This was accomplished by freezing the DNA at 0°C. p
B. subtilis strain BR151, which harbors UB110, was transformed by ligating DNA, plated on Luria broth supplemented with 1.1% agar and 3% skim milk, and incubated at 37°C. After 16 hours, a lawn of transformants was observed directly on the transfer plate. 10-1
There are approximately 2,000 colonies on the dilution plate, 1
There were approximately 240 colonies on the 0-2 plate. A milk clarification zone was observed after 17 hours. Colonies from the clear zone were selected and streaked for single colonies. After 24 hours, all plates were cleared with protease produced by the recipient strain. A single colony was isolated and grown for 5 hours at 37°C in Psnassay broth with 10 ug/ml chloramphenicol.

Example II Plasmids pGx2109, pGX2110 and pG
Isolation and Identification of X2111 The medium was centrifuged and isolated from the cell pellet by a modified boil preparation method as follows. Cells in 8% sucrose, 5% Triton, 50mM EDTA, 50mM Tris
Resuspend in 350ul of pH 8.0 (STET) and
00ug lysozyme was added and the suspension was incubated at 37°C for 15 minutes and then boiled for 1 minute. Chromosome DN
A and proteins were removed by centrifugation at 12,000xG for 20 minutes and added to proteinase in 5ul (50ug/ml).
) was added to the pellet and incubated for 20 minutes at 37°C. Samples were heated at 70° C. for 20 minutes to inactivate proteinase K. To precipitate the DNA, add 2 volumes of Inprovil alcohol to vinegar and keep the solution at -20°C.
I left it for 30 minutes. The pellets were washed with 70% ethyl alcohol and dried under vacuum. plasmid DNA
was dissolved in 50 ut of distilled water, and Bacillus subtilis (B, sub-t
5 ul was used to transform the .ilis) (BR151).

Transformants were screened for kanamycin and chloramphenicol resistance and milk clearance. The transformed colonies were either kanamycin resistant, chloramphenicol resistant and protease positive or kanamycin resistant, chloramphenicol resistant and protease negative. No protease-positive chloramphenicol-sensitive colonies were observed. Nine independent clones were identified.

Nine clones were single colonies purified by streaking on Luria broth, 1.1% agar and 3% skim milk. Extracellular supernatants from each purified colony ij Vasantha and Fre@se.

J, Baet, 144:1119-1125 (1980
) was assayed for protease activity in the presence of EDTA or phenylmethylsulfonyl fluoride.

The protease activity of two colonies was reduced to 9 by EDTA.
5, and was not inhibited by PMSF. Seven colonies were inhibited by HPMSF (95%) and not inhibited by EDTA. The former was indicated to produce neutral protease, and the latter to produce alkaline protease.

Plasmids from each single colony were isolated in duplicate as described above. The isolated plasmids were of recognized restriction. Colonies producing neutral protease contained one plasmid, labeled pGX2109 with restriction sites as shown in Figure 2. This is USDA
Northern Regional Reae-a
rch Laboratory, Peoria, III
It was precipitated as B15436. Alkaline protease producing colonies contain two closely related plasmid tags pGX2110 and pGX2111. pGX2
The restriction site at 110 is illustrated in FIG. Plasmid pGX2111 contains Bg1II at the 3' end of the insert.
-p except that it contains an additional 600 base pair segment in the BamHI/MboI region.
The same restriction map was given to GX2110. Plasmid pGX2110 was precipitated at NRRL as B15437.

To identify the location of control regions and structural genes within the pGX210 insert, 8-base pair SalI linkers were introduced at various restriction sites and their effects on protease production determined. In different experiments, the plasmid was transformed with SmaI using the conditions specified by the manufacturer and also as directed by the manufacturer.
or Hlndlll, BglIII and treated with S1 nuclease. S
An alI linker was inserted into the cleavage site using T4 ligase. This plasmid-containing linker is Bacillus subtilis (B.
, subtilis) (Contents and Dub
nau, supra). The transformants were analyzed for protease activity due to their activity on clear skim milk. Sal at SmaI, BglII and HindIII sites
Introduction of the I linker did not inactivate the protease gene. The plasmid is then cleaved with ClaI and the large fragment circulated by ligation in the presence of T4 ligase and isolated from B. subtili.
s) was used to convert. Transformants were analyzed negative for protease activity. According to the results of these experiments, the ClaI site is apr[Bamp]
It is clear that there is something to do with genes. The amino acid sequence deduced from the DNA sequence showed broad homology to the published amino acid sequence of subtilisin (Figure 3), and the ClaI site was located in codon 36 of the mature protein.

Example III Overproduction of protease in B. subtilis culture Plasmid pGX2109 (neutral protease), pG
X2110 (alkaline protease) and pBD6
4 (B, the plasmid used to generate the Bacillus subtilis BR151 transformation vector) was transformed into Bacillus subtilis (B, s
ubtills). This transformant was also bred. The protease activity in the supernatant is
Measurements were made using Hyde Powder Azul as a substrate. B. subtilis transformed by pGX2109 and pGX2110 had approximately equal protease activity after 48 hours, and B. subtilis transformed by the live protease pB064.
ilis) is B. amyloliquefaciens
7 and B, subliti transformed by pGX2109 or pGX2110.
The activity was 1/100 of that of s.

Example ■B. Expression of protease of stage zero sporulation mutant strain of S. subtilis B, protease negative of S. subtilis, stage zero sporulation mutant strain n (strain name IS53.SpOOB)
was converted in the manner described above. Cells were plated on L, B, 1.1% agar and 3% skim milk. Milk clearance was determined by pGX in BR151 (spore-forming strain).
It was not recognized from 2110 onwards. A replicable plasmid expression is therefore a non-protease producing B, sub
tilis, especially those that do not normally produce spores. Naturally occurring stage zero sporulation,
subtills have not been found to produce proteases, and there is a correlation between the lack of sporulation and the lack of protease production.

Example V Construction of a Secretion Vector for Δ22-Protein A Using the apr[BamP] Signal Sequence Secretion Vector Using the apr[BamP] Signal Sequence The entire apr[BamP] gene was transferred to the plasmid pGX2.
M13 as an EcoRI-Sall fragment of 125
Phage vector M13mp9 (Messing and Viera, Gene, 19:269-276 [198
2]) branched within. Plasmid pGX2125 is
pGX2110-11 containing apr[BamP]
It was generated by subcloning the SmaI-BglII fragment into GX2104, where it resides on the EaoRI-SalI fragment. Noris e
tal.

Oligonucleotide-controlled mutagenesis methods (Nua, Acl
dsReg, 11:5103-5112 [1983]
), a Bamri I site was cleaved as usual by inserting the sequence GGATCC just before the union of the signal coding sequence and the proso-quence.

M13mp9 containing apr[BamP] was grown in YP broth and single strand phage DNA was isolated from Zoll.
er and Smith (supra).

This mutagenic oligonucleotide 5′TGCCCAGGCGGCAGGGGATCCGA
AATCAAACGGGGA3' and M13m9 Universal Malaimer 5'GTAAAACGACGGCC
AGT3' is the tensile DNA (0.8pmole
) for 20 pmol and 16 pmol, respectively.
Annealed at e. Annealing and elongation are described by Norris at al. It was carried out accordingly. After 2 hours of incubation at room temperature, this mutant mixture was digested with gcoRI and 5ail and ethanol using BeoRI and SalI digested pGX251 (shuttle vector) as carrier DNA. A subspecimen of was subjected to gel electrophoresis and the fragment of interest (EeoRI for SalI fragment
) was successfully synthesized in vitro. The remaining precipitated DNA was ligated and E. coli was transformed with the ligated DNA. Twenty-four E. coli transformants were screened for the presence of a BamH1 site, one of which was found to contain a BamH1 site. This phyllasmid is PGX2134 (3rd
(see figure), which is represented by Leoli and B,
Replication is possible in both subtilis. Plasmid pGX2134 can be used to insert an atypical or atypical gene with a BamHI site at the 5' end, directed to expression and secretion of the production of the atypical gene in B. subtilis.

Plasmid pGX2912 is derived from S. aursus (5th
Contains the full length genetic coding for protein A (see figure). Plasmid pGX291
2 was (partially) digested with BclI and pvuII.

This BelI-PvuII fragment contains amino acid 23 (Δ2
It contained the entire 3' end of the Protein A gene starting at the second codon (2-Protein A). The BclI sticky terminal is complementary to the sticky terminal created by BamHI cleavage.

Plasmid pGX2134 - BamHI and pvu
Digested by II. This BclI-PvuII fragment, encoded for Δ22-Protein A, was ligated into the cleaved pGX2134 and created the plasmid pGX21.
36 were produced. Grasmid pGX2136 is apr
(BamP) promoter and regulatory region, has a signal peptide coding sequence, has two codons,
C.C.A.

Fuses to Δ22-protein A via GGG, apr
Corresponds to the first two amino acids of the pro-region from [BamP]. apr[BamP]
The sequence of pGX2136 at the fusion portion of Δ2222 and Δ2222-Protein A coding can be expressed as follows.

apr [BamP], which specifies the genetic information of the promoter, signal sequence, professional region, and approximately 60 mature proteins;
E from plasmid pGX2109 referring to the gene part
The coRl-EcoRV fraction was cloned into the M13 phage vector M13mp19 at the BcoRI and Smal sites (J, Norraander, T, Kem
pe and J.

Messing, Gene, 26:101-106 (1
983:]). npr[BamP] (EciRI to E
Mi3mp19 containing coRV) fraction was very unstable. Therefore, independent plaques (~36) were screened for phages with the entire insert by agarose gel electrophoresis. Single-stranded DNA is created from this, and oligonucleotide-directed mutagenesis is performed using Norri.
The following was generated using the method of S et al.
ATCCTCAGCTTAA 3' (20 pmol) and M
13 universal primer (12 pmol) is the template DNA
A (0.8-1.0 pmol) was used. Mutagenesis occurred as outlined for apr[BamP]. E. coli was transformed, and 24 transformants were screened out. One of them has a BamHI site, and this Grasmid is designated as pGX2138 (see Figure 6), which is compatible with E. coli and B. coli. su
btills. Plasmid pGX2] 3B can be used to insert a similar gene with a RamHT site at the 5' end, and B.
directed to the production expression and secretion of heterologous genes in subtilsi.

Plasmid pGX2138 contains BamHI and EcoR
Digested by V. Plasmid pGX2912 containing the protein A gene was (partially) digested with BelI and EcoRV. The BclI-PvuII fragment with the coding for Δ22-Protein A was ligated to the cleaved pGX2138-DNA (EcoRV and PvuII cleavage, both resulting in blunt ends) to generate plasmid pGX2140 (see Figure 6). did. Plasmid pGX2140 contains the bromomotor and regulatory region of npr[BamP], the signal peptide coding sequence, and two codons corresponding to the first two amino acids of the front region of npr[BamP], GCT,
Contains GAGs.

Limited analysis of pGX2140 indicates that it is N-terminal (N t
The entire BclI protein A gene lacks 22 amino acids at the C-terminus and 45 amino acids at the C-terminus.
It showed that it was picking up pieces. npr[B
The sequence of pGX2140 at the junction of the [amP] and Δ22-protein coding sequences is apr[
BamP]/Δ22-Protein A fusion, the two amino acids are ala and glu at the junction.

Example ■ △22-Protein A expression and secretion apr[Bam
P] Δ22 in the control of promoter and regulatory region
- plasmid pG containing the prodin A coding sequence
X21.36, and Δ22-Protein A (under the control of the npr[BamP] promoter and regulatory region)
C-terminal amino acid 45 or less) p containing the coding sequence
Each of GX2140 was used to transform B. subtilis strain 4935 by the method of Chang et al., and transformants were selected for chloramphenicol resistance. B. subtllis strain 4935 is
B, a low protease-inducing strain of subtilsiBR151. All chloramphenicol anti-transformers were tested using an enzyme-linked immunoabsorption assay (enzyme-linked immunosorbent assay).
-linked immunosorbent as!
Iay) j' was found to be protein A positive.

There are two transformants carrying pGX2136 and pGX2140.
Three different media were each inoculated (see Table I) and allowed to grow for 24 hours. The amount of Δ22-protein secreted into the medium was determined by the method of Lofdhal et al. (Proc, Nat'1.Ac
ad, Sc1. .

80:697-701 [1983]). The results are shown in Table I. apr[BamP] and npr[
These results demonstrate that the promoter and regulatory region of both genes of B. subtilis (BamP) are capable of directing the expression and secretion of heterologous proteins in B. subtilis.

1) Per liter of medium A, tryptone 33g; enzyme extract 20g: NaCl 7.4g: 3M NaOH 12me;
Na2HPOq 8g: KH2PO44r; Kazaamino acids 20g: / Glucose 10g: MnCl 20.06mM
and an initial pH of 7.5.

2) Vasantha and Freese, J. Bacte
riol. , 144:1119-1125 (1980)
A synthetic medium with 100 mM MOPS + 1% glucose + 0.2 malic acid + 1% yeast extract + 1% casamino acids replaced with 100 mM potassium phosphate as described by.

Example VII Construction of a Secretion Vector for Prorennin Using the apr[banP] and npr[bamP] Signal Sequences Escherichia coli (E. coli) strain GX1670 was constructed using the plasmid p
GX2231, which is joined by a 10-amino acid spacer sequence and consists of a fusion of the first 27 amino acids of the trpB expression product and its 2N-terminal amino acids (
Δ2-prorennin) and carries a gene specifying the genetic information of the prorennin sequence linked by a 10-amino acid spacer sequence. Δ2-prorennin has been shown to have the biological activity of full-length prorennin, i.e., undergoes autocatalytic cleavage to create the active form of the milk clotting enzyme rennin (co-filed commonly assigned U.S. patent application No. 528,421, the disclosure of which is included in the cited document). Strain GX1670 was obtained using accession number NRRL B-15571.
A Northern Regional Research
ch Laboratory, Peoria Ill.
It was precipitated with inoi.

Plasmid pGX2231 was developed by Holmes and Quigl.
ey method (Anal. Biochem, 114:1
93-197 ([1981]), Escherichia coli (E, co
li) extracted from strain GX1670.

Using the method of Norris et al., supra, a BelI restriction site is introduced against the exact flow of the Δ2-prorennin coding region by inserting the following hexanucleotide TGA TCA: The Δ2-prorennin gene is cleaved with BclI and a restriction enzyme that produces a blunt end 3' to the gene's transcription terminator.

apr[Ba
Plasmid pGX2134 (Example V) containing the mP] gene is digested with BamHI and PvuII, and the small fractions are discarded. The fractions from the split pGx2134 are ligated to the Bcl) digested Δ2-prorennin gene. The resulting plasmid is -ala-gl
Δ2- by the sequence symbolizing y-asp-gln-
Contains the complete apr[BamP] signal peptide coding region structurally linked to the coding region for brolennin.

npr[ with the BamtLI m position inserted flush with the signal peptide coding sequence.
BamP]-containing plasmid pGX2138 (Example ■
) is digested by BamHI and EaoRV and both small fractions are discarded. Both large segments from pGX213B are ligated to the BclI-digested Δ2-prorennin gene created as described above. The resulting plasmid contains a complete npr[BamP] signal peptide-coding region structurally linked to the coding region for Δ2-prorennin by a sequence encoding ala-glu-asp-gin-.

apr[BamP]]/Δ2-glorenni and apr[B
amP]/Δ2-prorennin fusion was isolated from Bacillus subtilis (B.) by the method of Chang and Cohen, supra, respectively.

subtilis) strain BR151, and transformants are screened for chloramphenicol resistance. Chloramphenicol-resistant transformants are inoculated into culture medium and grown overnight. The presence of secreted proteins containing the Δ2-glolennin sequence is confirmed by Western analysis. Secreted proteins that can be recovered from the culture medium by conventional methods are autocatalytically cleaved and disclosed in U.S. Patent Application No. 52
Active rennin is produced by the method of No. 8,421.

[Brief explanation of drawings]

Figure 1 shows alkaline glothiase (apr [BamP
]) Shows a partial restriction map of plasmid pGX2110, generated by inserting into pB064 an insert encoding the promoter and regulatory regions as well as the associated structural genes. In Figure 1, ■ is apr(Bu
mP], ■ represents another B, amyloliquef
nciens DNA, - indicates pBD64 vector sequence. A detailed map of the insert showing some restriction sites and the location of apr[BamP] (→) is shown below the plasmid. Figure 2 shows B. amyloliquefaciens.
Neutral protease derived from npr[BamP]
Partial restriction map of plasmid pGX21090, generated by inserting into pBD64 an insert encoding the promoter and ligature regions as well as the associated structural genes for the plasmid pGX21090. In Figure 2, ■1 is npr[Ba
mP], ■ represents other B, amyloliquef
aciens, - indicates pBD64 vector sequence. Several restriction sites and -npr[BamP] (→)
A detailed restriction map of the insert showing the location is shown below the plasmid. FIG. 3 shows the encoded amino acid sequence of the apr[BarnP] gene and B. amyloliquefaciens
depicts the encoded amino acid sequence of alkaline propase (saftilisin) produced by. Both chains were sequenced from several independent overlapping clones. Putative liposome binding site and transcriptional terminator
is underlined. Area of inappropriate combination (
Amino acid sequences previously published (indicated in Figure 3 by an asterisk) (Smith) et al. BiolChem,,2
14:5974-5976 (1966]) is 56.57
(Pro, Asn): 61 (Asp); 88, 89 (S
er, Ala); 98, 99 (Aap, Ala): 1
58.159 (Ser, Thr): 25J, (Gim)
It is. FIG. 4 represents the nucleotide sequence of the npr[BamP] gene and the encoded amino acid sequence of the neutral protease produced by B. amyloliquefaciens. Both chains were sequenced from several independent overlapping clones. The putative liposome binding site and transcriptional terminator are underlined. The N-terminus of the mature protein is
) N- of neutral protease from NRRL B5411
It was confirmed that it completely matched the terminal sequence (Lev
y et al., Proc, Nat'l. Acad,Sei,,U
,S.A. 72:4341-4345 [1975)). Figure 5 shows protein A (S.
Figure 2 is a graphical illustration of the structure of plasmid pGX2136 containing the genetic coding portion for P. aureus) II. Figure 6 shows protein A (S. aureus [S,
Figure 2 is a graphical illustration of the structure of plasmid pGX2140, which contains the genetic coding portion for P. aureus. Patent applicant: Genex Corporation F/
5゜FIG 6゜Continued from page 1 Priority claim [Phase] June 8, 1984 [Phase] United States (US
) ■ 618 Procedural Amendment (Voluntary) 1.11159・11.8・”, 11 Yo 1 Commissioner of the Japan Patent Office 1, Indication of the case Japanese Patent Application No. 139173/1982 2゜ Name of the invention Microorganism capable of expressing protease enzyme Method of manufacturing: 3. Relationship with the case of the person making the amendment Patent applicant name Genex Corporation 4 Agent Postal code 105 Address 1-4-10-5 Nishi-Shinbashi, Minato-ku, Tokyo Subject of amendment (1) Name of the invention in the application Column (2) Column for the representative of the patent applicant in the application (3) Power of attorney and its translation (4) Specification 6, Supplement, E,) Contents JP-A-O-AEO-91980 (1)
9) (1) Amend the title of the invention in the application as shown in the attached sheet. (2) Amend the representative of the patent applicant in the application as shown in the attached sheet. (3) Supplement the power of attorney and its translation as shown in the attached document. (4) Engraving of the specification (changing the contents) Procedural amendment (method) % formula % 1. Indication of the case Japanese Patent Application No. 139173/1982 2. Name of the invention Manufacture of microorganisms for the expression of protease enzymes Method 3, Name of the person making the amendment Name: Genex Corporation 5, Date of amendment order: October 30, 1980 6, Drawing subject to amendment 7, Contents of amendment: Engraving of the drawing (no change in content)

Claims (1)

[Scope of Claims] 1. A DNA sequence consisting of (a) a replicon, (b) a promoter and a regulatory region that control the expression and secretion of protease in different Bacilli, and 2. A method for producing a microorganism capable of expressing a grotease enzyme by converting a Bacillus having a replicable plasmid expression vector comprising a DNA sequence encoding the amino acid sequence of a desired protease linked to the Bacillus. B. subtill
s) and the DN encoding the promoter and regulatory region
A sequence and protease are B, amylolique
Claim 1 derived from fa-ciens
The method described in section. 3. The method of claim 2, wherein the B. amyloliquefaciens protease is selected from neutral proteases and alkaline proteases. 4. The method according to claim 2, wherein the B. amyloliquefaciens protease is a neutral protease. 5. The method according to claim 2, wherein the B. amyloliquefacians protease is an alkaline protease. 6. Claim 2, wherein said B. subtilis comprises a homologous plasmid. Third. The method according to paragraphs 4 and 5. 7. (a) a replicon, (b) a DNA sequence consisting of a promoter and regulatory region controlling the expression and secretion of Bacillus protease, and (c) an amino acid sequence of a polypeptide operably linked to the aforementioned promoter and regulatory region. A replicable plasmid expression vector capable of directing high level expression and secretion of a polypeptide in a transformed Bacillus, comprising a DNA sequence encoding a polypeptide. 8. The replicable plasmid expression vector according to claim 7, which is a DNA sequence comprising a promoter and a regulatory region. 9. The propeptide is B, amyloliquefa
9. A replicable plasmid expression vector according to claim 8, which is a cien protease. 10, the polypeptide is B, amylolique
9. A replicable plasmid expression vector according to claim 8, which is a C. aciens neutral protease. 11. The common polypeptide is B, amylolique
9. A replicable plasmid expression vector according to claim 8, which is a C. aciens alkaline glothiase. 12, Plasmid pGX2109゜13, Plasmid pGX2110゜14, Grasmid pGX2111. 15. A microorganism transformed with the replicable plasmid expression vector of claim 7. 16, Claim No. 8. 9th. 10th or 1st
A microorganism transformed with the replicable plasmid expression vector of item 1. 17. Microorganism transformed by plasmid pGX2109 18. Microorganism transformed by plasmid pGX2110. 19, microorganism transformed by plasmid pGX2111. 20. The microorganism is Bacillus subtilis
Claim 15. 17th. The microorganism according to item 18 or 19. 21. The microorganism is Bacillus subtilis (B, subtili@)
The microorganism according to claim 16, which is 22, (a) (1) Replicon: (II) DNA sequence consisting of a promoter and regulatory region that controls protease expression and secretion in Bacillus, and Qli
(b) transforming a Bacillus with a replicable plasmid expression vector comprising a DNA sequence encoding the amino acid sequence of a heterologous polypeptide operatively linked to said promoter and regulatory regions; (6) recovering the polypeptide from the medium; and (6) recovering the polypeptide from the medium. 23, promoter and regulatory region are B, amyloliq
23. The method of claim 22, wherein the expression of protease of P. ue-faeiena is controlled. 24. The Bacillus to be converted is Bacillus subtilis (B.
24. The method of claim 23, wherein the method is: ls). 25, the polypeptide is B, amylollque
26. The method of claim 24, wherein the polypeptide is B. faciena protease.
25. The method of claim 24, which is P. faciena neutral protease. 27, the polypeptide is B, amylolique
25. The method of claim 24, wherein the method is a P. faciens alkaline protease. 25. The method of claim 24, wherein the replicable plasmid expression vector is plasmid pGX2109. 29. The method of claim 24, wherein the replicable plasmid expression vector is plasmid pGX2110. 30. The method of claim 24, wherein the replicable plasmid expression vector is plasmid pGX2111. 31. Promoter and regulatory region of genes selected from apr[BamP] and apr[BamP], which are structurally fused with a structural gene encoding a polypeptide other than B and amylollquefaelens protease to the 3' end of the signal peptide coding sequence A replicable plasmid expression vector consisting of. 32. The replicable plasmid expression vector according to claim 31, wherein the structural gene fused to the promoter and the regulatory region consists of a gene encoding a protein with prorennin activity. 33. The replicable plasmid expression vector according to claim 32, wherein the protein having prorennin activity is Δ2-glorennin. 34. The replicable plasmid expression vector according to claim 31, wherein the structural gene fused to the promoter and the regulatory region consists of a gene encoding a protein with protein A activity. 35. The protein with protein A activity is Δ22-protein A
35. The replicable plasmid expression vector of claim 34. 36, Claim No. 31. 32nd. 33rd゜34th
Alternatively, microorganisms of the genus and species of Bacillus subtilis transformed by the replicable plasmid expression vector of paragraph 35. 37. Promoters and regulatory regions of genes selected from apr(BamP) and npr[BamP] and apr
A vector consisting of a replicable plasmid containing a sequence of deoxynucleotides flanking the 3' end of a signal peptide coding site, consisting of an endonuclease restriction site not naturally present in [BamP] or apr[BamP]. 38. The vector of claim 37, wherein the promoter and regulatory region are from the apr[BamP] gene. 39. The vector of claim 38, wherein the endonuclease restriction site adjacent to the 3' end of the signal peptide coding sequence is a BamH1 site. 40. Plasmid pGX2134°41, the vector according to claim 37, wherein the promoter and regulatory region are from npr (BamP). 42. Endonuclease adjacent to the 3' end of the signal peptide coding sequence. The vector according to claim 41, wherein the restriction site is the tmHI site. 43. Plasmid pGX2138°