JPS63290899A - Fragment of human igefc protein and production thereof - Google Patents

Abstract

NEW MATERIAL:A protein consisting of a linker peptide and fragment of protein from 226th amino acid to 365-400th amino acid of human IgE protein bonded to the C end of the linker peptide. USE:A remedy for allergy. PREPARATION:For example, a conventional plasmid pGETtrp302 constructed so as to express Fc domain of human IgE is cut with a restriction enzyme and the resultant cohesive end is filled with Escherichia coli DNA polymerase I large fragment and then EcoRI linker is linked with T4 DNA ligase and linkers of both ends are cut and DNA is relinked to afford a plasmid, which is then inserted into Escherichia coli to transform the bacterium to recombinant Escherichia coli having ability capable of producing fragment of linker peptide and human IgEFc and the recombinant Escherichia coli is cultivated to provide the fragment of human IgEFc protein.

Description

[Detailed Description of the Invention] The present invention discloses human IgE (
The present invention relates to a protein containing a fragment of human immunoglobulin E) Fc protein, and a method for producing the same.

Problems that will be resolved in the future F present on membranes of cells and basophils
It binds to c receptors. Fa, which is part of the Y-shaped branch
b releases biologically active substances such as histamine from those cells by reacting with antigens, causing immediate allergies, etc. On the other hand, Fc part CCH2 does not have a Fab part and consists only of an Fc part. °C:l+3.
CH4) has the ability to bind to Fc receptors, so a complete I
It competes with gE in binding to Fc receptors, and since it does not have a Fab portion, it does not react with antigens, so it inhibits the release of histamine, etc. from cells, and as a result, it can cause immediate allergies. It is known that there is no

On the other hand, human IgE, which is the material for Fc, exists in extremely small amounts in normal human serum; it is prepared from myeloma patient serum, which contains a high concentration of IgE, and from time to time, standard specimens of the same species are used. It is impossible to obtain any desired amount of human IgE. Some of the present inventors have attempted to obtain a large amount of highly purified human IgE from the culture supernatant by culturing established human myeloma cell lines. Opened (Japanese Patent Application Laid-Open No. 58-96028)
However, it is difficult to obtain the Fc portion by digesting IgE with a proteolytic enzyme, and only an extremely small amount of FcL can be obtained, so this method has not yet become a practical method.

Recent advances in molecular biology have shown that D encoding the Fc portion
It has become possible to introduce NA into bacteria and express it. By applying this genetic recombination technology, Fc DNA
If Fc protein can be expressed in bacteria, it will be possible to prepare large quantities of Fc protein, paving the way for its practical use as an allergy therapeutic.

The base sequence of the IgE gene of myeloma cells established from myeloma patient ND was determined [Procedure of the National Academy of Sciences (Proc. N.
atl, Acad, Sci, U, S, A,)79666
1 (1982), Nucleitsk Acids Research (
Nucleic＾cids Res,)dan719(19
83)), the expression of Fc fragments in Escherichia coli has been reported [Nuclear Aids Research (Nu
Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci.)

However, since Fc proteins have nine cysteine residues per single chain, Fc proteins extracted from E. coli cells under reducing conditions have the correct concentration. Not building the next structure.

The technology for assembling proteins produced using recombinant DNA technology into the correct higher-order structure has not yet been established and remains a challenge for the future.

One method for solving the above problems is to use animal cells as hosts and secrete proteins to the outside of the cells. IL-2
A technology has been completed to express human IgEFc protein DNA in animal cells using the leader sequence of Fc protein and efficiently secrete and accumulate Fc protein in the culture medium [Patent Application No. 61-182]
No. 457], and a technology for efficiently isolating and purifying Fc proteins accumulated in the culture medium has also been completed (Patent Application No. 61-28).
No. 1871).

The present inventors conducted research to enable the expression of Fc protein in E. coli, and succeeded in using the method described above in the animal cells [encoding the entire Fc portion (amino acid 2''@ to amino acid 1@) of human IgE protein. However, it was not possible to obtain an Fc protein that showed strong reactivity with anti-human IgE antibodies.

In order to solve the 4 points, the present inventors proposed that if a recombinant that modifies Fc[white[DNA and expresses a fragment favorable to E. coli] could be obtained, it would be possible to prepare a large amount of modified fragments; Focusing on the fact that this substance is expected to make a great contribution to the treatment of allergies, we created a recombinant E. coli strain transformed with DNA that has a base sequence encoding a partially missing Fc protein. When the protein was produced by culturing, a protein was found that strongly reacted with anti-human IgE antibodies. Specifically, DNA corresponding to the carboxy-terminal 76 amino acid residues of Fc protein
Recombinant E. coli 294/pG that does not encode
E Ttrp712 (having DNA encoding amino acid 22' to amino acid 4#0) (see Figure 1 for amino acid numbers) [hybridoma±,
47 (1985)) was found to react strongly with the human IgEI kit (IgE Reagio Tsugi (Shionogi & Co., Ltd.)).That is, the above recombinant bacterial cells were cultured and the protein was injected into the bacterial cells. This product was extracted using an extract containing guanidine hydrochloride, and then subjected to a purification process including immunoaffinity chromatography, which revealed that human IgE
The linker peptide: Asp"' -Asn ---Arg -Ala", a part of the Fc protein of human IgE, is removed at the C-terminus of Met-Leu.
' was obtained. Although the above recombinant E. coli contains DNA encoding the amino acids up to Asp"'-Pro"', Asp"@---Ala"' has some amino acids deleted on the C-terminal side.
They produced a new protein containing .

In this way, the present inventors have discovered a linker peptide in which not all but part of the Fc protein of human IgE is bound to the C-terminal side, specifically, 22
The present invention was achieved based on the discovery that a novel protein in which a fragment starting from the 6th amino acid and ending with the 365th to 400th amino acids is bound exhibits strong reactivity with anti-human IgE antibodies.

That is, the present invention provides (1) a linker peptide and its C
Protein (1) consisting of a fragment starting from amino acid 226 of human IgE protein bound to the end and ending with amino acid M at positions 365 to 400 (see Figure 1)
, (2) A method for producing the protein, which comprises culturing a recombinant E. coli cell having the ability to produce ffl white matter (1) and collecting the protein accumulated in the culture, (3)
The protein production method of (2) above, in which the protein is collected by a method including immunoaffinity chromatography;
4) The present invention relates to a method for producing the protein described in (3) above, which uses a monoclonal antibody in immunoaffinity chromatography.

The protein (1) of the present invention is a novel substance, and such a protein has never been obtained before.1 The protein (1) of the present invention is attached to the N-terminal side of a fragment of human IgEFc protein. Any linker peptide may be used as long as the protein is smoothly expressed in E. coli, but specifically, Met-L
Examples include things like su.

Examples of recombinant E. coli cells having the ability to produce the protein (1) of the present invention include the aforementioned E. coli 294/p GETtr
A specific example is p712, but the present invention is not limited thereto.

For example, D having a base sequence encoding protein (1)
A recombinant E. coli cell transformed with NA and producing protein (1), or a base sequence encoding a linker peptide and a base encoding a part of human IgE protein linked to its 3'-end. D having the array
Any E. coli cells transformed with NA, such as recombinant E. coli cells that produce protein 'Jt (1) through processing within E. coli, may be used.

As a method for producing the protein (1) of the present invention.

In addition to culturing recombinant E. coli cells capable of producing the above protein (1) and purifying the product, human IgEF bound to a linker peptide and its C-terminus.

A fragment having a desired amino acid can also be obtained by excising amino acids from the C-terminal side of a protein or a fragment thereof.

Note that in the protein of the present invention, even if some amino acids are deleted or substituted with other amino acids, those having the same effect as the present invention are included in the protein of the present invention.

Culture of transformed E. coli cells used in the present invention.

Medium 1 known per se, for example in M-9 medium, 15-40°C,
Preferably at 24-37°C for 3-48 hours, preferably 5
This is carried out for up to 12 hours, and aeration and stirring may be added if necessary.

After culturing, the cells are collected by a known method, suspended in a buffer containing a protein denaturant such as urea or guanidine hydrochloride, stirred in a cool place, and then centrifuged to collect the supernatant containing the protein. How to obtain, or suspend in buffer and sonicate,
After destroying the bacterial cells with lysozyme and/or freezing and thawing, a method of obtaining a supernatant containing the protein by centrifugation can be used as appropriate. Add liquid and stir (0~
A preferred method is to obtain a supernatant by centrifugation after 0.5 to 8 hours at 10°C.

The Fc protein fragments can be separated and purified from the above extract by appropriately combining known separation and purification methods. That is, for example, separation and purification methods such as immunoaffinity chromatography using monoclonal antibodies, gel filtration chromatography, and ion exchange chromatography can be appropriately selected and used in combination. It is best to use them in order.

Although an anti-human IgE polyclonal antibody can be used as a ligand for immunoaffinity chromatography, it is particularly preferable to use a monoclonal antibody that recognizes the Fc portion.

E23 as a monoclonal antibody that recognizes the Fc portion
5 I 63. Examples include E 120.02 [hybridoma 4, 47 (1985)], and E 120.02 is particularly preferred.

As a carrier for immunoaffinity chromatography, Affigel-10 (Bio-Rad), CNBr-activated Sepharose 4B (Pharmacia), Formylcellulofine (Seikagaku Corporation), etc. are used, and Affigel-10 is particularly preferred.

Fc? To purify If white matter fragments by immunoaffinity chromatography using E 120.02 as a ligand, the extract containing the pre-Fc protein fragments is first thoroughly dialyzed against a buffer solution (such as a phosphate buffer); The supernatant from which insoluble matter has been removed by centrifugation is adsorbed onto the column equilibrated with the buffer and eluted with the buffer. Appropriate amounts of protein denaturants (urea, etc.) can be added to the buffer solution, and the type and concentration of these additives can be adjusted.
The pH of the buffers can be combined and used as a suitable eluent. An ultrafiltration membrane or the like can be used to concentrate both the obtained Fc protein fragments.

Gel filtration chromatography, for example Sephacryl 5
- It is carried out by applying known means using a zoo (Pharmasha) column.

Ion exchange chromatography is carried out by applying known means, for example, using a DEAE-Toyovar 650M (Toyo Soda Kogyo) column.

To measure the purity (specific activity) of the human IgEFc protein fragment thus produced, a commercially available human IgE measurement kit, such as IgE Riajiotsugi (Jiotsugi Pharmaceutical Co., Ltd.), is used.
This can be done using

According to the production method of the present invention, it is possible to obtain a highly purified human IgEFc protein fragment-containing protein that can be used as a pharmaceutical or the like.

For example, by purifying an Fc protein fragment-containing protein produced by an E. coli cell transformed with a DNA having a base sequence encoding a part of human IgEFc protein by the method of the present invention, a protein having the following properties can be obtained. A purified human IgEFc protein fragment-containing protein can be obtained.

(1) SDS-polyacrylamide gel electrophoresis (under reducing conditions) shows two bands, and the measured molecular weights are 20,000±1,000 Daltons and 18,000±1,000 Daltons, respectively.

(2) It has methionine (or its formyl derivative) as the amino terminal amino acid.

(3) It has alanine as the carboxy-terminal amino acid.

(4) It has a specific activity of IXIO' units/■ or more.

In the present invention, the activity (specific activity) of Fc protein fragment-containing protein for determination of protein purity is measured using IgE
The test was carried out by radioimmunoassay using a Riagiotsugi kit.

The protein of the present invention has low toxicity and has a therapeutic effect on allergies caused by IgE.

Therefore, the protein of the present invention can be used as a therapeutic agent for allergies such as allergic rhinitis, atopic dermatitis, and bronchial asthma. For example, spray as a spray solution, ointment, cream, etc.
It is administered parenterally by application or orally as a tablet. The dosage varies depending on the nature and route of administration, but is approximately 10 ng to 100 μg per dose, and is administered approximately three times a day.

In the specification and drawings of this application, when bases, amino acids, etc. are indicated by abbreviations, IUPAC-IUB
Comm1ssion on Bioche+aica
It is based on the Nomancla-ture abbreviations or common abbreviations in the field, examples of which are listed below.

Furthermore, if an amino acid has optical isomers, unless otherwise specified, the L-isomer is assumed to be indicated.

DNA Deoxyribonucleic acid A Adenine T Thymine G Guanine C Cytosine SDS Sodium dodecyl sulfate Gly Glycine Ala Alanine Val Valine Leu Leucine 11a Isoleucine Ser Serine Thr Threonine Cys Cystine 1/2 Cys Half cystine Met Methionine Glu Glutamate A g p Aspartate Lys Lysine A r g Arginine His Histidine Phe Phenylalanine Tyr Tyrosine Trp Tryptophan Pro Proline Asn Asparagine Gln Glutamine The present invention will be explained in more detail with reference examples and examples below, but the present invention is not limited to these. do not have.

In addition, p G E T trp described in Reference Example 2 below
E. coli 294 (I F 0-1417
1) by Cohen et al. [Proceedings of the National Academy of Sciences]
Knee J (Proc, Natl.

Acad, Sci, U, S, ^,)61.2110(1
The transformant E. coli 294/pG E Ttrp302 obtained by the method described in 972) is
Institute of Fermentation (IFO) L: IFO-14
285 To L Te Yori Ifc! retail.

Furthermore, p G E Ttrp7 also described in Reference Example 2
E. coli 294 (IF 0-14171
) in Cohen et al.'s Proceedings of the National Academy of Sciences (Proc, Natl, Acad, Sci, U, S
, A. ) 61.2110 (1972), the transformant E. coli 294/p obtained by transformation.
G E Ttrp712 was developed by the Fermentation Research Institute (IF).
O) as IFO-14609, and since May 21, 1988, the Microbial Research Institute (FRI) of the Ministry of International Trade and Industry.
It has been deposited as FERM P-9382.

Reference Example I Preparation of E 120.02 Afuigel 10 column Anti-human IgE monoclonal antibody, E 120.0
2 (Hybridoma, Sat., 47 (1985)) 360
PBS containing s+g (137mM NaCl, 2, 7m
M KCI, 8.1mM Na, HPO, 1.5m
MK1. PO,, p) 17.4) 75ml of solution was added to 2Q of 0. IM Na+lCO.

After dialysis against the solution overnight at 4°C, Affigel 10
(Bio-Rad) Add to 75 mΩ and mix at 5°C.
Shake for several days. The mixture was transferred onto a glass filter, and the gel was washed with PBS to remove proteins that did not bind to Affigel 10. The binding rate to the monoclonal antibody in this operation was approximately 87%, and approximately 4.2 g of monoclonal antibody was bound to E120.02-g per 101 mQ of 7 figs.
0 gels obtained from Affigel 1075mQ were mixed with PBS,
3M urea, 0.2M sodium acetate buffer (pH 4,0) containing 0.15M NaCl, 0.15M Na
After sequentially washing with 0.2M acetic acid containing 1:1 and PBS, it was packed into a column with an inner diameter of 2.61 to produce an E120.02-Affigel IO column.

Reference Example 2 Construction of carboxy-terminally deleted Fc protein expression plasmid p G E Ttrp712 C of human IgE
, - Plasmid pG E Ttrp30 constructed so that the C4 region polypeptide is expressed in E. coli
2 [Kurokawa, et al., Nucleic Acids Research,
) Dan, 3077-3085 (1983); Japanese Patent Application Publication No. 1983
-44399 publication] with restriction enzyme MstII,
After filling the resulting sticky ends with E. coli DNA polymerase large fragment.

The Ec*RI linker containing the translation stop codon phosphorylated at the 5' end of 50nH-dCTAGAATTCTAG to T
4DNA ligase (New England Biol
ABS) was used for binding. After part of the linker bound to both ends was cut with EcoRI, the DNA was recombined to construct a plasmid pGETtrp712 encoding up to the 480th amino acid of human IgE protein. Using this plasmid, the method of Cohen et al. (Proc, Natl, Acad, Sci, U, S
Transformant E. coli 294/pG E Ttrp71 was obtained by transforming E. coli 294 (IFO-14171) according to A.) 69.2110 (1972) J.
2 (see Figure 2).

Example 1 Culture of transformant Transformant E. coli! Bacterium 294/ pG E Ttrp7
12 (see Figure 2) in 1.0% glucose, 0.2%
After culturing in M-9 medium containing casamino acids at 37°C for 6 hours, the bacterial cells were collected.

Example 2 Extraction from Bacterial Cells 65 g of cryopreserved microbial cells obtained by culturing according to the method described in Example 1 and freezing at -20°C were treated with 7M guanidine hydrochloride.

PBS containing 20mM 2-mercaptoethanol
(pH 7.4) The mixture was suspended at 200 mA and stirred at 5°C for 4 hours.

PB 8267■Q was added to this suspension and stirring was continued for an additional hour, followed by centrifugation at 30,100×g for 1 hour to obtain a supernatant, which was then dialyzed against PBS 4Ω at 5°C for 2 days. . In addition, PBS was replaced three times during the test.

The dialyzed fluid was centrifuged and the supernatant 4'lOwa Q (2
115B; 98.7 x 10' units) was obtained.

Example 3 Production of Fc protein fragment ■ Immunoaffinity chromatography E 120.02-Affigel 10 column prepared in Reference Example 1 (2,6X
13.6m) was equilibrated with PBS, a portion of 15 fujl of the dialyzed fluid obtained in Example 2 was applied to the same column, and then 1,000 mQ of the same buffer and 0.15 M NaCQ were added.
0.2M sodium acetate buffer (pH 7,2) containing 6
0.2M sodium acetate buffer (pH 5,0) containing 0mQ, 3M urea, 0.15M NaCQ 1
It was washed sequentially with 00mQ and PBS 200Ω. 3
The active fraction eluted with a buffer containing M urea is about 80m Q
was dialyzed against PBS2Q for 24 hours at 5°C. In addition,
I replaced the PBS once during the trip. The dialyzed fluid obtained through three chromatography operations was centrifuged to obtain a supernatant 24
1■Q (31,3+*g; 55,4 x 10" units) was obtained.

(2) Gel filtration chromatography using Sephacryl S-200 The centrifuged supernatant obtained in (1) was concentrated using YM-5 Menpuran (manufactured by Amicon, USA).

The entire amount of the obtained concentrate was applied to a Sephacryl S-200 column (1.6×105 m) equilibrated with PBS in advance, and the Fc protein fragment was eluted using the same buffer. Active fraction 24.7mA (9.4mg; 19.
3×101 units) were obtained.

■ The active fraction obtained by ion exchange chromatography using DEAE-Toyopearl 650 M was dissolved in 10 mM Tris-HCQ.
The mixture was dialyzed against 500 mQ (pH 8°2) buffer for 24 hours. The buffer solution was exchanged once during the process. After centrifuging the dialysate, the supernatant was preliminarily purified with lomM Tri
s-tic! DEAE-Toyopearl 650M column (0.72X17) equilibrated with 1 (pH 8.2) buffer.
cm) and washed with the same buffer solution. Proteins bound to the column were mixed with 10 mM Tris-HCQ (pH 8
, 2) Elution was performed using a linear NaCQ concentration gradient gradient in which 100 mΩ of the same buffer containing 0.3 M NaCQ was continuously added to 100 mQ of the buffer. NaCQ concentration 14
The high specific activity fraction eluted around 0■H was 17.2iQ (1
, 8 mg; 6-5 x 10' units) were collected.

The substantially pure Fc protein fragment produced by the above method had the following properties.

(1) Unity: The purity of the Fc protein fragment obtained in Example 3 was determined by the Laemmli method [Nature J
As a result of 5DS-polyacrylamide slab gel electrophoresis carried out according to the method of Fc protein fragments under reducing conditions, the Fc protein fragment mainly showed two bands (see Fig. 3).

(2) Molecular weight: The molecular weight of the Fc\X white matter fragment was determined by 5DS-polyacrylamide slab gel electrophoresis to be 2 under reducing conditions.
0,000±1,000 and 18,000±i,
It was calculated as ooo Dalton.

(3) Amino acid composition: Fc protein fragment 15.6 obtained in Example 3
Transfer μg to a glass hydrolysis test tube and add constant boiling point hydrochloric acid containing 4% thioglycolic acid.

After sealing the tube under reduced pressure, it was hydrolyzed at 110° C. for 24,48,72 hours. After hydrolysis, the tube was opened, hydrochloric acid was removed under reduced pressure, and the residue was dissolved in 0.02N hydrochloric acid.
Amino acid analysis was performed using a model amino acid analyzer. Cystine and cysteine were determined using the Haas method [Methods in Enzymol].
After oxidizing the Fc protein fragment with performic acid, it was hydrolyzed in constant boiling point hydrochloric acid under reduced pressure for 24 hours, and quantified as cystic acid using an amino acid analyzer.

Amino acid analysis values were determined by averaging the values obtained for 24.48 and 72 hours of hydrolysis. However, the values of serine and threonine were determined by extrapolating the hydrolysis time to 0 hours. The results are shown in Table 1.

Table 1 Amino acids Mol% Asp/Asn 9.8Thr
13.5Ser
9.4Glu/Gln
11.3Pro 3.8
aty 7.2Ala
4.91/2Cys
3.2V a l
5,6Met 1.6I
le 3.4Lau
9.8Tyr2.7 Phe 3.3L
ys4.0 His 2・4A
rg, 2.4Tr
p 1.5(4)NH
, terminal amino acid sequence: 24 μg of the Fc protein fragment obtained in Example 3
by applying the automated Edman degradation method using a gas phase protein sequencer (Model 470A, Applied Biosystems, USA).

The NH2-terminal amino acid sequence was analyzed. Phenylthiohydantoin amino acid (PTH = amino acid) was identified by high performance liquid chromatography using a Micropac C18-3 column (Parian, USA). PTH-amino acids detected in each step are shown in Table 2.

Table 2 Step Detected PTH-Amino Acid 1
Methionine 2 Leucine 3 Aspartic acid 4 Asparagine 5 Lysine 6 Threonine 7 Phenylalanine 8
Serine 9 Valine 10 X Umbrella 11 Serine 12 Arginine 13 Aspartic acid 14
Phenylalanine 15 Threonine skewer: Unable to identify (5) COOH-terminal amino acid 90 μg of Fc protein fragment obtained in Example 3
was placed in a glass hydrazine decomposition test tube, anhydrous hydrazine O, 1rnQ was added, and the tube was sealed under reduced pressure.
Heated at 0°C for 6 hours. The obtained hydrazine decomposition product was dried to solidity using a speed-vac consedator (manufactured by Servant, USA), and then dissolved in distilled water. Benzaldehyde was added to this solution, stirred at room temperature for 1 hour, and centrifuged to obtain a supernatant. The supernatant was dried and subjected to amino acid analysis using a Hitachi fIB835 amino acid analyzer. As a result, 1. In mole alanine was detected.

The human IgEFc protein fragment-containing protein produced according to the present invention is a new substance useful in the treatment of allergies caused by IgE, and the present invention also enables mass production of the protein, which is suitable for industrial use. The above usefulness is great.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the nucleotide sequence of the linker and IgE Fc portion contained in plasmid PGETtrp712 translated into an amino acid sequence. Figure 2 shows the plasmid PGETtrp used in the present invention.
712 horizontal construction plan. FIG. 3 shows the results of 5DS-polyacrylamide slab gel electrophoresis of the protein obtained according to the present invention.

Claims (4)

[Claims] (1) Starting from the 226th amino acid of the human IgE protein linked to the linker peptide and its C-terminus, 36
A protein consisting of a fragment ending at amino acids 5 to 400. (2) Starting from the 226th amino acid of the human IgE protein linked to the linker peptide and its C-terminus, 36
A method for producing the protein, which comprises culturing a recombinant E. coli cell capable of producing a protein consisting of a fragment ending with amino acids 5 to 400, and collecting the protein accumulated in the culture. (3) The method for producing a protein according to claim 2, wherein the protein is collected by a method including immunoaffinity chromatography. (4) A method for producing a protein according to claim 3, which uses a monoclonal antibody in immunoaffinity chromatography.