JPH01153093A - Genetic dna participating in production of fc binding protein - Google Patents

Abstract

PURPOSE:To enable mass production of a protein A, by finding out a genetic DNA capable of producing a protein having the ability to bind to the Fc part of immunoglobulin G with Escherichia coli, Bacillus subtilis, yeast, etc., which are nonpathogenic germs. CONSTITUTION:A genetic DNA having about 1.05kb molecular size, (A) one recognition site by restriction enzymes ClaI, ScaI, SalI, Sau3A, EcoRI, SacI and AccI and (B) no cleavage site by restriction enzymes BglII, EcoRV, StuI, PvuII, HpaI, XhoI, BclI, KpnI and Sph is used to transform Escherichia coli, Bacillus subtilis, yeast, etc., which are nonpathogenic germs. Thereby a protein A, capable of specifically binding to the Fc part of immunoglobulin G and especially useful in the field of purifying monoclonal and polyclonal antibodies, clinical diagnostic agent, immunological biochemistry, etc., can be mass-produced.

Description

[Detailed Description of the Invention] The present invention relates to immunoglobulin G (hereinafter referred to as r1gGJ).
(7) It relates to genetic DNA involved in the production of proteins that have the ability to bind to the Fc portion.

Conventionally, substances that specifically bind to the IgG(7) Fc portion, particularly protein A, have been widely used in the purification of monoclonal and polyclonal antibodies, application to clinical diagnostic agents, and the field of immunobiochemistry. In addition, for application in the pharmaceutical field, antigen-antibody complexes are removed and treated using the so-called plasma perfusion method, in which plasma from patients with immune disorders or cancer is passed through a column bound to an insoluble carrier. It is used in research that is expected to be effective [for example, cancer
) + measurement.

675 (1980)).

This protein A is produced by Staphylococcus aureus (
It was isolated and purified from the cell wall of Cowan I strain using the enzyme lysostaphin. However, recently, genetic DNA involved in the production of protein A or its related proteins has been cloned, and a method for producing protein A and its related proteins using recombinant vectors carrying these and transformants using the vectors has been developed. has been published (for example, JP-A-5
No. 9-113890, No. 59-205996, and Japanese Patent Publication No. 59-501693-1 No. 60-500480).

On the other hand, the present inventors are also researching an efficient method for producing such active protein, and have been conducting research on protein 8, which has the same effect as protein 8.
Discovered and disclosed a method for secreting and producing RP-2 substance outside of bacterial cells (Patent Specification No. 191648/1981 or Collection of Lecture Abstracts at the 1988 Japan Society for Fermentation Engineering, No. 133)
(see page).

According to the above-mentioned conventional technology, antibody purification, rgG adsorption removal,
Although it is possible to produce the protein A active substance 5RP-2 useful in immunobiochemistry, the cultivation must use the pathogenic bacterium Staphylococcus aureus. To achieve this, culture tanks and purification equipment designed to prevent viable bacterial cells from being released into the environment are required, and special care must be taken to ensure that toxins derived from Staphylococcus are not mixed into the product. , it is difficult to supply 5RP-2 substance at low cost.

Therefore, the present inventors conducted research on the cloning of genes involved in the production of 5RP-2 substances independently of the preparation of known protein A-producing genes, and found that non-pathogenic bacteria such as Escherichia coli, Bacillus subtilis, yeast, etc. They discovered a genetic DNA capable of expressing this trait and completed the present invention.

. That is, the present invention involves the production of a protein that has the ability to bind to the immunoglobulin G(7) Fc portion, has a molecular size of about 1.05 kb, and has the following properties: Showing the following sensitivity to restriction enzymes, namely (a) C1a I,S
ca I, Sal I, 5au3A, EcoRI
, there is one recognition site by Sac T and Acc I, (b) BgIll, EcoRV, StuI,
PvuI, HpaI, XhoI.

The present invention provides genetic DNA characterized in that it is not cleaved by Bcl I, Kpn I and sph.

In addition, immunoglobulin G (IgG) (7
) The Fc portion is derived from a mammalian animal such as a human, monkey, rabbit, guinea pig, dog, pig, mouse, etc.
It refers to a region other than the Fab region that includes the variable region (V domain) involved in antigen binding of IgG, which is a type of well-known antibody, and which binds to the cell membrane [e.g.
1881 (1979)], such Fc
The protein that has the ability to bind to the protein is not particularly limited as long as it is a protein-like substance that is known per se, but more specifically, the protein that was discovered by the present inventor and named 5RP-2. The following properties were determined: A) Molecular weight 24,000 B) Isoelectric point pH 4.5 C) Ultraviolet absorption Maximum absorption at 275 nm (E I cm
= 1.60) D) Human 1gG binding ability 11■/■
Proteins that exhibit the following can be mentioned.

Therefore, the present invention provides at least genetic DNA involved in the production of 5RP-2.

The DNA was obtained from the central laboratory of Sanraku Co., Ltd., the applicant of the present application, since the secretion-producing strain of 5RP-2 is a mutant strain of Staphylococcus aureus, a protein A-producing strain.
It can be carried out by the following method using the cells of the SR-1 strain stored and maintained at the Minami 4-9-1) of Gerozawa City under storage number B-1964.

(i) Cells are lysed by the lytic enzyme lysostaphin.C
, R. Wilson, P.A., Totsuten and J.N.
, Parduin, Applied and Environmental Microbiology (Appl, Environm, Mi
crobiol).

Co., 368 (1981)), and total DNA is extracted. (ii) Next, this DNA was digested with restriction enzyme 5au3A.
The DNA fragments were partially degraded by agarose gel electrophoresis to obtain purified DNA fragments of 2 to 10 kb. (iii
) On the other hand, E. coli plasmid vector pUc8 CJ,
Vieira and J, Mesothing: Gene
,■.

259 (1982)) was digested with the restriction enzyme BamHI, mixed with the previously prepared 5au3A fragment of the 5R-1 strain, and ligated with the DNA ligation enzyme T4 ligase. (iv) The thus obtained recombinant plasmid was injected into host Escherichia coli E and coli JM103 strains using a known method [Ko, Mandel and A, Higa: Journal Opmolecular Biology (J, Mol, Biol, ) 53.1
59 (1970)), and colonies harboring the plasmid were selected using a selection medium containing ampicillin (35 μg/ml).

Colonies having the desired protein A activity were selected from among the above-mentioned format-transformed colonies by the following method. LB = A nitrocellulose membrane is placed on an agar medium (1% batato tryptone, 0.5% yeast extract, Na (11.0%, agar 1.5%)), and a cellulose acetate membrane is placed on top of that. 100-15 per petri dish on top of this
Spread 1 ml of bacterial suspension Q containing the transformant diluted so that 0 colonies grow. This is 37°
C. After 24 to 48 hours of incubation, the nitrocellulose membrane was removed and soaked in 1% gelatin solution for blocking).
．． Wash with 05M phosphate buffer. Next, it is soaked in rabbit serum for 2 hours and washed. Next, it was immersed in a solution of peroxidase-labeled anti-rabbit immunoglobulin G (goat-derived) and incubated at 37°C for 2 hours.
The area around the colony containing the 5RP-2 producing gene turns blue. The colonies thus obtained are isolated in pure form,
H, C, Vernpoim and J.
Dolly's method [Nukreitsk Acid Research (N
ucleic acids research), 7
．． 1513 (1979)) and digested with various restriction enzymes and their 5RP-
Gene DNA of interest can be prepared to confirm the productivity of the two substances.

More specifically, it is a transformant in which the periphery of the colony containing the above-mentioned 5RP-2 producing gene develops a blue color, and was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry on December S, 1985. Escherichia collie (Escherichia collie) was deposited and given the deposit number 9746.
richia codan) 7-3 by the above-mentioned known plasmid extraction method and various restriction enzyme treatments.

In addition, the above-mentioned Escherichia codan (hereinafter referred to as r
The restriction enzyme map of plasmid 5RP-1 containing the gene DNA of the present invention, which is carried by E. coli J) 7-3, is shown in Figure 1 fb).
The restriction enzyme map is shown in Figure 1 (al).

As will be described later, the genetic DNA of the present invention has the ability to cause the transformant to express the ability to produce protein A-like protein by transforming various host bacteria with a recombinant plasmid containing it. and is effective for increasing the production of the protein.

The present invention will be explained in more detail with reference to the following examples, but it goes without saying that the present invention is not limited in any way based on these descriptions.

Example 1 Staphylococcus aureus strain SR-1 (see above) Tryptone soy broth medium (50%
d/250mi Erlenmeyer flask) and inoculated at 37℃ for 2 hours.
The culture was carried out with shaking for 0 hours. The bacterial cells are collected by centrifugation,
It was washed once with 0.05M Tris buffer (pH 7.5) containing 0.15M NaCl.

The bacterial cells were again collected by centrifugation, suspended in 5 ml of the same Tris buffer, and treated with the lytic enzyme lysostaphin (10 ml).
20 μl of 2/ml) was added. This is 37'C Ni30
After holding for a minute, pronase F (20nw/mj Q,
5 ml was added and the reaction was further carried out at 37°C for 260 minutes. From the bacterial cell lysate obtained in this way, Qigyo and Miura's method
chim, Biophys, Acta) 72.619
(1963)) to purify chromosomal DNA. Solution containing chromosomal DNA (0,034μg/μJ) 160μ
restriction enzyme 5au3A I (2,000 units/
-) 0.5 μn and the buffer specified by the manufacturer (Wako Pure Chemical Industries) were added (total volume 200 μj2), and the reaction was carried out at 37°C for 5 minutes, followed by 100 μn of phenol! was added and extracted. Furthermore, 100 μl of phenol-chloroform (1;1) was added for extraction, and twice the amount of ethanol was added to the aqueous layer for 120 μl.
The DNA was precipitated by standing at °C overnight.

Collect the precipitate by centrifugation and TE buffer (10mM
Tris Tris buffer EDTA, pH 8,0) 20
Dissolved in 0 μl.

On the other hand, plasmid pUc8 (J, Wieira and J,
Menoshing: Gene, 19.259 (
1982)) was completely digested with the restriction enzyme Bam1l I and treated with calf alkaline phosphatase (3.99μ).
(total volume: 50 μm) containing 1 μl of T4 ligase (1,000 units/-), pH 7.
, 6 of 40mM Tris-HClCl (MgC
j! z 20mM, dithioerythritol 20mM
, ATP 1.2mM) was added and the reaction was carried out at 10°C for 20 hours. Add 2 times the amount of ethanol and heat at -20°C.
After standing overnight, the precipitate was collected by centrifugation and dissolved in 50 μl of TE buffer, which was used as a DNA solution for transformation.

Using 10 μl of this DNA solution, use a known method.

Mandel and A, Higa: Journal of Molecular Biology (J, Mo1. Biol,) 53.159 (
E. coli JM103 (manufactured by Pharmacia Japan) was transformed with E. coli JM103 (manufactured by Pharmacia Japan Co., Ltd.) using (1970)), and a Shiichi agar medium containing ampicillin (35 μg/d) (1% Batato tryptone, 0.5% yeast extract, NaCj!0.5) was used. %
, agar 1.5%, pH 7.0) (37°C, 24°C).
Transformants were selected by (time). Two membranes, a nitrocellulose membrane and a cellulose acetate membrane, were placed in this order on a separately prepared agar medium, and a growing colony of the previous transformant (ampicillin-resistant strain) was transferred onto these membranes by the replica method.

Bacteria growing on the replica membrane of the 5RP-2 colony were removed by removing the cellulose acetate membrane, and then the nitrocellulose membrane was removed. The nitrocellulose membrane was briefly immersed in a 1% gelatin solution dissolved in 0.05 M phosphate buffer (pH 7.5 for blocking) and then washed with 0.05 M phosphate buffer.

Next, the membrane was washed by immersing it in rabbit serum (1,000-fold dilution) for 2 hours. Finally, it was immersed in a peroxidase-labeled anti-rabbit IgG (goat) (20,000-fold dilution) solution and allowed to react at 37'°C for 2 hours. A total of about 30,000 colonies were examined using the method described above, and 5 colonies were found to be blue in color, indicating that they were protein A productive. Among them, strain 7-3 which showed a particularly clear blue color was isolated, and the plasmid was extracted and pSRP-1 was digested with various restriction enzymes. As a result, the following results were obtained.

(Recombinant plasmid (psRP) carried by strain 117-3
-1) is 5.9 kb, and the number of cleavages by various restriction enzymes is as follows.

Prison ndI[[4 Pstl 2 Sac I 2 Sail 2 Sma I I C1a I I EcoRI I Mlu I l 5car I Bgln 0 Bcl I 0 EcoRV 0 Hpa I 0 Kpn I 0 Pvu II 0 5p h I 0 5tu I 0 Xba I 0 Xho T O The restriction enzyme map of pSRP-1 is shown in FIG. 1(b).

(2) Insertion D derived from strain 5R-1 in pSRP-1
The NA fragment has a size of approximately 3.2 kb and is indicated by the thick line in FIG. 1(b) above.

Plasmid pSRP-1 of I゛・/NA''- in 5RP-2 was completely digested with EcoRI, and twice the amount of ethanol was added to the reaction mixture, and the mixture was incubated at -20°C.

- After standing overnight, the precipitate was collected by centrifugation, dissolved in distilled water, and dissolved in distilled water using a known method (B. Vuais et al.: Journal of Biol Chemistry (J. Biol. Chem.).

243, 4543 (1968)). This reaction product was used to transform E. coli J old strain 03, and colonies exhibiting protein A activity were searched for using the method described above, but none were found among the ampicillin-resistant transformants, and the plasmid was extracted. When the size of the molecule was examined, it was found that a 1.5 kb EcoRI-digested fragment of pSRP-1 was deleted.

Next, plasmid pSRP-1 was completely digested with Pstl, the DNA fragment was purified using the same method as above, and then treated with T44 ligase to transform E. coli JM103, and the protein A activity of the transformant was examined. All colonies showed activity, and when their plasmids were examined, it was found that they showed protein activity even though the 0.7 kb Pst1 degradation fragment was deleted.

Next, plasmid pSRP-1 was completely digested with Sal I, and the digested fragment was purified in the same manner as above and treated with T41J gauze. When we used this to transform E.coli JM103 and examined the protein A activity of the transformants, some showed strong activity and others showed weak activity, and the former was the same as the original pSRP-1. The latter was found to lack the 1.3 kb Sal 1 degradation fragment.

Next, ρ5RP-1 was completely degraded with old ndII, and the degraded fragment was purified and subjected to T4 ligase reaction in the same manner as above.

This reaction product was used to transform E. coli JM103, colonies with protein A activity were selected from among the transformants, and the plasmid was examined.
1lindI of b [[It has a degraded fragment and is 0.5 kb
It was found that the HindlI fragment was deleted.

Furthermore, pSRP-1 was completely digested with I'lsp T and subjected to 1.0% low melting point agarose gel electrophoresis.
The Msp I-digested DNA fragment of kb was purified. On the other hand, plasmid pUc8 was completely digested with Acc I and purified by ethanol precipitation, and then mixed with a 2.5 kb Msp I fragment and subjected to a T4 ligase reaction. This reaction product was used to transform E. coli JM103, colonies exhibiting protein A activity were isolated from the transformants, the plasmid of the strain was extracted, and the inserted DNA fragment was examined.2. It contained a 5 kb Msp I-digested DNA fragment. Finally, pSRP-1 was digested with Sac I, purified by ethanol precipitation, and then completely digested with Ps. The degraded product was separated by 1.0% low melting point agarose gel electrophoresis, and the 1.2 kb Sac I-Pstr
The fragment was purified. This includes plasmid pUC
18 was similarly digested with Sac I and Pst I, and after T4 ligase reaction, E. coli JM103
was transformed and examined for colonies with protein A activity, but no colonies were detected.

From the above results, the NA region essential for the production of 5RP-2 is approximately 1.05 kbD of the degradation product of Msp I and Sac I.
It is clear that it is in the NA SR region. This genetic DNA (1.05 kb) can be prepared as follows.

7-3 strain by known methods (11, c, Burnpoim and J, Dolly: Nucleic Ac1d Re5e).
Plasmid pSRP-1 extracted according to the method of Arch. This reaction product was subjected to 1.0% agarose electrophoresis, and a DNA fragment appearing at a position of approximately 1.05 kb in molecular size was extracted from the agarose using a known method (methodin enzymes).
Methods, inEnzymo, 1ogy) 6
8. p176 (1979)), the NA fragment essential for 5RP-2 production can be obtained.

The NA region essential for the production of 5RP-2 is shown in Figure 1 (a).
Indicated by the thick line.

Example 2 E. coli 7 3 strains (FERM P-9745)
Bacterial cells cultured on ordinary agar medium - Platinum loops were cultured on LB medium (Bactodrypton 1%, yeast extract 0.5%, NaCj! 1
%, ampicillin 50pg/d, 50+d/250mf
The cells were inoculated into f1 (erlenmeyer flask) and cultured at 37°C for 24 hours. This 0.1- was inoculated into 10 Erlenmeyer flasks containing 50- of the same medium, and cultured with shaking (rotary 220 rpm) at 37°C for 20 hours. Centrifuge the bacterial cells (8,0
00rpn+ for 15 min) and 501n1 of Tris buffer (Tris H (425mM, EDT)).
A 10mM, pH 8.0) suspended in shirizozyme
0 mg was added, reacted at 20° C. for 1 hour, and further subjected to ultrasonication (20 klLz, output 60 W) for 10 minutes to lyse the bacteria.

Adjust the pH to 3.7 with hydrochloric acid, let stand at room temperature for 20 minutes, and then centrifuge (8.00 rpm, 15 minutes) to remove supernatant liquid 4.
Got 5-. This was adjusted to pH 7.5 with IN NaOH, 2.7 g of NaCl was added, and the active fraction was separated by hydrophobic chromatography. After lyophilization, 5i p
It was dissolved in H7,51-Lis buffer (0.05M) and purified by gel filtration (Sephadex G-100).

After dialysis, freeze-drying was performed to obtain 4 mg of white powder (hereinafter referred to as 7-3 substance).

(i) SDS-Polyacrylamide snow #ΦLaemm
The method of Li et al. [Nature, 2
5DS-polyacrylamide gel electrophoresis was performed in accordance with 27° 1970). 7-3 after staining with Coomasible G250 using a white matter molecular weight marker (manufactured by Sigma)
The molecular weight of the substance was measured to be 24,000.

(ii) Etc. 1st 11 Urinary System Relsner et al.'s method [Analytical Biochemistry (Anal, Biochem,) 64.509
(1975)), and after electrophoresis, the gel was placed on an agarose plate supplemented with 1% pig serum for 20 minutes.
After being left for 2 hours at °C, the position of the 7-3 substance was detected by the sedimentation zone and compared with the isoelectric point marker. As a result, the isocenter of this substance was determined to be pH 4.5 to 4.8.

(iii) When the Kurigaishya Somoto substance was dissolved in distilled water and its ultraviolet absorption was examined,
The absorption peak is at 275 nm, and at this wavelength E,
cffi=2.0.

The absorption spectrum is shown in Figure 2.

(iv) Atrocious JJL 7-3 substance 6N) IC/! After decomposition at 1110°C for 24 hours, amino acid analysis was performed using an automatic amino acid analyzer (Hitachi model 835). The results are shown in Table 1.

Table 1 Amino acid composition Amino acids Number of residues Aspartic acid 45 Threonine 2 Serine 15 Glutamic acid 43 Glycine 5 Alanine 22 Valine 2 Isoleucine 9 0 Isine 23 Tyrosine 4 Phenylalanine 12 Lysine 21 Histidine 2 Arginine 3 Proline 12 Methionine 2 (v) H41 Taka Veracar method (W, Becker: Immunochem, 6.539 (19
When the binding ability with human immunoglobulin G was examined by 69)), it was found that this substance had the ability to bind with 11 μ of large immunoglobulin G per 1 μ of this substance. The binding strength of protein A tested at the same time was 8■.

From the above results, the protein A active substance produced by 7-3 (pSRP-1 retained) is the protein A active substance produced by 5RP-2 (patent application 1986-
No. 191648).

[Brief explanation of the drawing]

The bold line in Figure 1 fal is the restriction enzyme map of the genetic DNA of the present invention, (b) is the restriction enzyme map of plasmid 5RP-1 containing the genetic DNA of the present invention, and Figure 2 is the restriction enzyme map of the genetic DNA of the present invention. The ultraviolet absorption spectra of proteins produced by genetic DNA are shown. Patent applicant (191) Sanraku Co., Ltd.

Claims (1)

[Claims] 1. Immunoglobulin G(7) involved in the production of a protein having the ability to bind to the Fc portion, and having a molecular size of approximately 1.05
kb and exhibits the following sensitivity to the following restriction enzymes: (a) Cla I, Sca I, Sal I, Sau3A
, EcoR I , Sac I and Acc I have one recognition site, and (b) BglII, EcoRV, St
uI, PvuII, HpaI, XhoI, BclI,
Genetic DNA characterized in that it is not cleaved by Kpn I and Sph.