Vector for expresii and secretion of human interferon alpha(hifn), an escherichia coli strain for expression and secretion hifn(options) and the method of production of hifn

 

The invention relates to the field of biotechnology, particularly to the production of recombinant human interferon. Expressing the vector for the secretory production of human interferon alpha (hIFNcontains polynucleotide encoding a modified signal sequence of thermostable enterotoxin II E. coli, and polynucleotide encoding hIFN, legirovannye with his 3-end. The vector used in the method of secretory production of human interferon by transformation of E. coli strain and cultivation of the specified strain. The method allows to secrete soluble form of the active hIFNthat do not contain additional methioninamide residue at its N end, periplasm cells of E. coli. 8 C. and 5 C.p. f-crystals, 5 Il., 1 PL.

The present invention relates to expressing vector for secretory production of human interferon alpha (hIFNcontaining polynucleotide encoding a modified signal sequence of thermostable enterotoxin II E. coli, and hIFN- codereuse vector; and to a method of production of hIFNthat do not contain additional methioninamide residue at its N end and greater in periplasm E. coli cells.

Prior art

In 1957, Isaac and Lindenman reported that chickens infected with influenza virus And was producyrovtsa factor inhibiting replication of the virus, called interferon (Issacs, K. & Lindenmann, J. Proc.R.Soc.Lond., 147:258-267, 1957).

Human interferons are proteins, cytokines, inhibiting the immune response in vivo or replication of the virus, and are classified as interferon alpha (IFN), interferon beta (IFN) and interferon gamma (IFN) the type producing their cells (Kirchner, H. et al., Tex.Rep.Biol.Med., 41:89-93, 1981; Stanton, G. J. et al., Those. Rep.Biol.Med. 41:84-88, 1981).

It is well known that these interferons, acting together, have a synergistic effect, which manifests itself in antiviral activity, antitumor activity, activation of NK (natural killer cell) and in activity aimed at the inhibition of proliferation of bone marrow cells (Klimpel et al., J. Immunol. 129:76-78, 1982; Fleischmann, W. R. et al., J. Natl. Cancer. Inst. 65:863-966, 1980; Weigent, et al., Infect. Immun. 40:35-38, 1980). In addition, interferons act as the named action.

IFNproduced when the leukocyte stimulated b-cell mitogen, virus or cancer cells. Currently known genes encoding more than 20 types of interferons, each of which includes 165 or 166 amino acids.

IFNused for early clinical tests, obtained from leukocyte film, stimulated with Sendai virus, and its purity is only less than 1% (Cantell, & Hirvonen, Those. Rep. Biol. Med. 35:138-144, 1997).

In 1980, using techniques of recombinant DNA became possible to produce a large number of IFNa with biophysical activity (Goedell, D. V. et al., Nature, 287:411-416, 1980). Clinical tests using recombinant hIFNhas shown that it is effective for the treatment of various solid cancers, particularly bladder cancer, kidney cancer, HIV-associated Kaposi's sarcoma, etc., (Torti, F. M. J. Clin. Oncol. 6:476-483, 1988; Vugrin, D. et al., Cancer. Treat. Rep. 69:817-820, 1985; Rios, A. et al., J. Clin. Oncol. 3:506-512, 1985). It is also effective for the treatment of hepatitis C virus (Davis, G. G. et al., N. Engl. J. Med., 321:1501-1506, 1989) and limits its use as a therapeutic agent are expanding day by day.

Results cloning of the gene IFNis a mixture of proteins of a particular subtype, with similar patterns. Proteins of this subtype were named IFN-1, 2, 3, etc., Nature, 290:20-26, 1981).

Among several types of interferons hIFNisolated from human leukocytes, has a molecular mass of 17500-21000 and very high natural activity of approximately 2108u/mg protein. Jn vivo, IFNis a protein consisting of 165 amino acids. If the 23rd amino acid is lysine, the interferon is indicated IFN-2a (SEQ ID No:1), and if the 23rd amino acid is arginine, he indicated IFN-2b (SEQ ID No:2). First hIFNwas produced using cell culture. However, this method proved to be unsuitable for industrial production because of its low productivity, which is about 250 μg/L.

To solve this problem have been developed methods for isolating a large number altoadige time.

The most widely used is the method using E. coli, which is in accordance with the properties of E. coli cells producing IFNconsisting of 166 or 167 amino acids. These products contain additional meinenemy residue added at the N-Terminus due to the ATG codon present in the website of the initiating codon. However, it was reported that in the case of human growth hormone this additional meinenemy residue may stimulate unwanted immune response (patent publication EP No. 256843).

In addition, most downregulation of IFNaccumulates in the cytoplasm in the form of insoluble Taurus on and should be turned into active form in the re-laying the cleaning process. Because this method of re-laying is inefficient, IFNis partially restored in the form or forms an intermolecular disulfide-binding body or defective disulfide-linked body. Removal of by-products that cause significantly low yield, presents certain difficulties. In particular, it is extremely difficult to remove such undesirable side products interverterbral a foreign protein in a microbial cell, there were several attempts to develop a method, based on the efficient secretion of the soluble form of the target protein that does not have at its N-Terminus an additional methionine.

In this way the desired protein is expressed in the form of a hybrid protein carrying a signal peptide attached to the N-end. With the passage of the specified hybrid protein through the cell membrane signal peptide is removed by the enzyme in E. coli, and the desired protein is secreted in native form.

The way secretory production is more preferable than the method of microbial production, because the amino acid sequence and tertiary structure of the protein produced, usually, identical to the specified sequence and structure of the wild-type protein. However, the output, which gives way to the secretory production, in most cases is very low due to the lack of effectiveness as membrane transport and subsequent treatment. This corresponds to the well-known fact that the yield of protein of a mammal produced secretory way in prokaryotes, much lower than the output prokaryotic protein produced in the same way in prokaryotes. Were therefore take action to avoid the awning Korea No. 93/1387 describes the attempts of large-scale production of IFNusing the signal peptide of alkaline phosphatase of E. coli, but its release into the culture medium at 109IU/l (10 µg/l of culture medium) was very low. Therefore, development of a method of producing a soluble IFNnot containing at its N-end additional methioninamide balance, using a microorganism for large-scale production, is of great interest.

Previously the authors of the present invention was produced a new signal peptide thermostable enterotoxin II E. coli (patent application Korea No. 98-38061 and 99-27418) and it was found that this new secretory signal peptide can be used for large-scale reproduction of natural forms IFN. Namely, the authors of the present invention was constructed expressing the vector containing the gene, obtained by ligating IFN-coding gene, instead of the gene encoding enterotoxin II, with modified secretory signal peptide of E. coli, and was designed the way secretory production of IFNpossessing natural biological activity, involving the use of mi is the dominant vector.

Brief description of the invention

In accordance with the present invention is to obtain expressing vector that is capable of secretory production of human interferon alpha (hIFN).

Another objective of the present invention to provide a microorganism transformed by the specified expressing vector.

Another objective of the present invention is a method of producing a soluble form of hIFNusing the specified microorganism that does not contain additional methioninamide residue attached to the amino end.

Brief description of the graphical material

The above and other objectives and features of the present invention will be apparent from the following description of the present invention and the accompanying graphic material, which respectively illustrates:

Fig.1: the procedure for constructing vector pT-IFN-2a;

Fig.2: the procedure for constructing vector pT14SI-2a;

Fig.3: the procedure for constructing vector pT14SSI-2a;

Fig.4: procedure for constructing vector pT14OSSI-2a, and the purity of IFN-2a, expressed from recombinant cell lines, and the result of Western blot analysis, which confirmed the molecular mass of the expressed IFN-2b, respectively.

Detailed description of the present invention

In accordance with one of its aspects the present invention relates to expressing vector for secretory production of hIFNcontaining polynucleotide encoding a modified signal sequence of thermostable enterotoxin II (hereafter referred to as “mutant STII) and polynucleotide encoding hIFN, legirovannye with its 3'-end.

Polynucleotides, encoding hIFNand used to construct expressing vector of the present invention may be any of polynucleotides encoding arbitrary subtypes hIFNsuch as natural hIFN-2a (SEQ ID No:1), IFN-2b (SEQ ID No:2), IFN-1 and IFN-3, and they can also be a recombinant polypeptide, which has a modified base sequence is inficirovannye signal sequence of thermostable enterotoxin II E. coli of the present invention, legirovannoi before the 5'-end of polynucleotide encoding hIFNand used for secretory production of hIFNmay be polynucleotide encoding a mutant obtained by replacing one or more amino acids of the signal sequence of thermostable enterotoxin II E. coli described in SEQ ID No:3, and preferably one or more of its 4-th, 20-th and 22nd amino acids of the other(they) amino acid(s). Examples of such polynucleotides are polynucleotides, encoding the mutants obtained by the following substitutions: 4th amino acid threonine ([Thr4]STII); 4th amino acids threonine and 22nd amino acids-glutamine, respectively ([Thr4, Gln22]STII); 4th amino acids threonine, 20th amino acid with valine and 22nd amino acids-glutamine, respectively ([Thr4, Val20, Gln22]STII); and 4 amino acids threonine and 20 amino acids-valine, respectively ([Thr4, Val20]STII) signal sequence thermostable enterotoxin II E. coli (STII) described in SEQ ID No:3, a preferred polynucleotide sequences are SEQ ID nos: 4, 5, 6 and 7. However, it is known that due to the degeneracy of codon may exist some d is hydrated by introducing preferred E. coli codons without any substitutions in the amino acid sequence, can be used to stimulate the rate of expression of IFN.

In addition expressing vector of the present invention may also include the Shine-dalgarno sequence thermostable enterotoxin II E. coli (SD sequence, SEQ ID No:8) or its mutant sequence, legirovannoi before the 5'-end of polynucleotide encoding a modified signal sequence of thermostable enterotoxin II. Compared with the sequence of the wild type, which has 7 bases (TGATTTT) following the GAGG on the 5'-end sequence SD thermostable enterotoxin II in E. coli, is presented in SEQ ID No:8, mutant SD sequence has a shorter sequence of 6 or 5 bases. The use of this mutant may lead to an increase in the rate of secretion and the expression of IFN. However, if the specified sequence of bases becomes shorter by 4 bases, then the rate expression is markedly reduced. A specific example of a preferred mutant, which can be used in the present invention is a mutant SD sequence thermostable enterotoxin II E. coli having the nucleotide pokemania, can be any promoter that can Express the heterologous protein in a microorganism host. In particular, if the heterologous protein is expressed in E. coli, the preferred promoter is the lac promoter, Tac and arabinose.

The present invention also relates to transformed microorganisms, which can be obtained by transformation of these strains of E. coli, as BL21 (D3) E. coli (Novagen, USA) or XL-1 blue E. coli (Novagen, USA) using the specified expressiruemogo vector. Examples of the present invention are transformed microorganisms, as BL21(DE3)/pT14OSSI-2A-4T (“NM 10603”) E. coli BL21(DE3)/ pT14OSSI-2a-4T22Q (“HM 10611”) E. coli BL21 (D3) /pT14OSSI-2b-4T (“HM 10703”) E. coli BL21(D3)/pT14OSSI-2b-4T22Q (“HM 10711”) of E. coli.

The above transformed microorganisms have been deposited at the Korean center of cultures of microorganisms (cssm) (Address: Yurium Bidg., 361-221, Hongje 1-dong, Seodaemun-gu, Seoul 120-091, Republic of Korea) December 23, 1999, under access numbers xsm-10175, xsm-10176, xsm-10177 and xsm-10178 respectively, under the Budapest Treaty, concluded in accordance with the International agreement on the Deposit of microorganisms for the conducted method of production of hIFNthat do not contain additional methioninamide residue at its N-Terminus, by its secretion in periplasm E. coli, where the method involves culturing the transformed microorganism in a suitable culture conditions, which may be the same as the standard cultivation conditions used for the transformed microorganisms.

hIFNproduced secretory method of the present invention includes arbitrary subtypes hIFNsuch as IFN-1, IFN-3, etc. as well as natural subtypes hIFN-2a (SEQ ID No:1) and hIFN-2b (SEQ ID No:2) consisting of 165 amino acids. Furthermore, the method of the present invention can also be used for producing any other interferon, such as hIFNand hIFN.

In accordance with the method of the present invention is 80% or more IFNproduced by transformants E. coli present invention is secreted into periplasm with a high degree of productivity, making up more than 1 g/l Produced IFNa has the same amino acid p is minamikata, attached to its N end, and which detects biological activity similar to the activity of native IFN.

The present invention is illustrated in detail by the following examples which do not limit its scope. Comparative example: gene IFN-2a and construction of a vector containing this gene.

The gene encoding hIFN-2a, was obtained by PCR using human genomic DNA as template and SEQ ID No: 10 and 11 as primers. Primer SEQ ID No:10 was designed to obtain a NdeI restriction site (5'-CATATG-3'), located above the codon encoding the first amino acid (cysteine) natural hIFNand primer SEQ ID No:11 was designed to obtain a BamHI restriction site (5'-GGATCC-3'), located downstream from the termination codon of natural hIFN.

Amplificatory PCR product was digested with the enzymes NdeI and BamHI to obtain a DNA fragment encoding hIFN-2a. This DNA fragment was integrated into the NdeI/BamHI site of the vector pet-14b (Novagen, USA) to obtain the vector pT-IFN-2a.

In Fig.1 illustrates the above Procera, containing the signal sequence enterotoxin genes and IFN-2a

To obtain gene signal sequence enterotoxin II E. coli, a pair of complementary oligonucleotides SEQ ID No:12 and 13 were designed based on previously known nucleotide sequence of the signal peptide of enterotoxin II E. coli and synthesized using a DNA synthesizer (Model 380B, Applied Biosystem, USA). The above oligonucleotides were designed to embed a BspHI restriction site (site complementary NdeI restriction site) above the initiation codon of enterotoxin II E. coli and MluI restriction site introduced by silent substitutions in the other end. Both of the oligonucleotide was annealed at 95°C To produce a blunt on the ends of the DNA fragment having a nucleotide sequence encoding a signal sequence enterotoxin II E. coli. The above DNA fragment was integrated into the SmaI site of the vector pUC19 (BioLabs, USA) to obtain the vector pUC19ST.

In addition, the vector pT-IFN-2a, containing the gene for IFN-2a, obtained in the comparative example was subjected to PCR using primers SEQ ID No:14 and 15 for ligating signal peptide intertax is on IFN-2a, and primer SEQ ID No: 15 was designed to BamHI restriction site (5'-GGATCC-3') was introduced following the termination codon of the gene. The DNA fragment containing the specified polynucleotide encoding of natural IFN-2a, amplified by PCR using the above-mentioned polynucleotide primers. Amplificatory DNA fragment was digested with enzymes MluI and BamHI to obtain a DNA fragment of IFN-2a with MluI/BamHI-ends.

The vector pUC19ST containing the signal peptide of enterotoxin, were digested with enzyme MluI, and then hydrolyzed BamHI to obtain a vector fragment with MluI/BamHI-ends. The specified portion of the vector ligated with a DNA fragment IFNa-2a, resulting received vector pUC19SIFN-2a.

Vector pUC19SIFN-2a were digested with enzymes BspHI and BamHI to obtain a DNA fragment (564 p. N.). The DNA fragment was incorporated into NcoI/BamHI-site of the vector pET-14b (Novagen, USA) to obtain the vector pT14SI-2a.

In Fig.2 illustrates the above procedure for constructing vector pT14SI-2a.

Then strain BL21(DE3) E. coli was treated with 70 mm solution of calcium chloride with obtaining competent E. coli, a ZAT the respective IFN-2a, selected the standard method based on the sensitivity of the transformed vector to antibiotics, and meant 10600 NM E. coli.

In addition, the vector pT14SI-2a was subjected to PCR using primers SEQ ID No: 16 and 17 for amplifying the DNA fragment obtained by sequential ligation sequence Shine-Dalgarno enterotoxin; signal peptide of enterotoxin; and gene IFN-2a, and then the DNA fragment was digested with the enzymes XbaI and BamHI to obtain the inserts.

The resulting insert ligated into XbaI/BamHI-site of the vector pET-14b (Novagen, USA) to construct a vector pT14SSI-2a.

In Fig.3 illustrates the above procedure for constructing vector pT13SSI-2a. Strain BL21(DE3) E. coli (Stratagene, USA) was transformed with vector pT14SSI-2a with getting transformant denoted by NM 10601 E. coli.

Comparative example 2: Construction of a vector containing the signal sequence enterotoxin genes and IFN-2b

23rd lysine codon of the gene IFN-2a in the vector pT14SSI-2a was replaced by arginine codon by site-rather IFN gene-2b. Vector pT14SSI-2a were subjected to hybridization with synthetic oligonucleotides SEQ ID No:19 and 20, containing the replaced codon, with the formation of hybrid molecules was carried out by amplification of DNA using pfu DNA polymerase (Stratagene, USA) and four nucleotidase (ATP, GTP, TTP, P), which prolong these oligonucleotides in the direction 5'3'.

The sequence of interferon-2b

Amplificatory DNA fragment was isolated and thereto was added restricteduse enzyme DpnI to remove the untransformed plasmids.

XL-1 Blue E. coli (Novagen, USA) transformed specified a modified plasmid. Determined the sequence of bases of DNA isolated from transformed colonies, and thus obtained plasmid pT14SSI2b, which contained the gene encoding arginine instead 23rd aminosilane IFN-2a, i.e. lysine.

Then BL21(DE3) E. coli transformed by the modified vector pT14SSI-2b with getting transformant denoted by NM 10701 E. coli by the method described in comparative example 1. Analysis of N-terminal aminates the transformant expressed IFN2b, having a native amino acid sequence.

Example 1: Construction of a vector containing the mutant signal peptide of enterotoxin

(1) Construction of vector containing [Thr4]STII

For modification of specific amino acid residue signal peptide sequence enterotoxin received vector containing polynucleotide encoding mutant signal sequence enterotoxin, using site-directed mutagenesis described below.

First, the vector pT14SSI-2a, obtained as described in comparative example 1 was subjected to PCR using oligonucleotides SEQ ID No:22 and 23, as a result, in accordance with the procedure site-directed mutagenesis described in comparative example 2 was obtained a modified plasmid, where the 4th amino acid of the signal sequence enterotoxin replaced by threonine (Thr).

XL-1 Blue E. coli (Novagen, USA) transformed specified a modified plasmid. Determined the sequence of bases of DNA isolated from transformed colonies, and thus obtained plasmid, which contained the gene encoding the signal peptide of placentas is Lyali enzymes XbaI and MluI, and then built into the XbaI/MluI-plot vector pT14SSI-2a with obtaining vector pT14SSI-2a-4T.

Then BL21(DE3) E. coli (Stratagene, USA) was transformed with vector pT14SSI-2a-4T with getting transformant E. coli, denoted by NM 10602 E. coli.

Vector pT14SSIa-2a-4T designed using pT14SSI2b, and then in accordance with the method described above, transformed into BL21(DE3) E. coli (Stratagene, USA) to produce transformant E. coli, denoted by NM 10702 E. coli.

(2) Construction of a vector containing [Thr4,Gln22]STII

Vector pT14SSI-2a-4T, obtained in stage (1), was subjected to PCR using oligonucleotides SEQ ID No: 25 and 26, which were designed so that in it the 22nd amino acid signal peptide of enterotoxin with Thr in the 4th position was replaced by a Gln codon in accordance with the procedure site-directed mutagenesis in stage (1), resulting in the received modified plasmid.

Then XL-1 Blue E. coli (Novagen, USA) transformed specified a modified plasmid. Determined the sequence of bases of DNA isolated from transformed colonies, and thus the floor is Oksana, with Thr and Gln in the 4th and 22nd amino acid positions of the signal sequence enterotoxin respectively. Then BL21(DE3) E. Coli (Stratagene, USA) was transformed with vector pT14SSI-2a-4T22Q in accordance with the method described in stage (1), the result obtained transformant Escherichia coli, designated NM 10604 E. coli.

To modify the sequence Shine-Dalgarno modified signal sequence enterotoxin with the formation of the sequence SEQ ID No:9 vectors pT14SSI-2a-4T and pT14SSI-2a-4T22Q was subjected to the procedure site-directed mutagenesis described in stage (2) using the oligonucleotides SEQ ID No:27 and 28 to obtain the desired modified plasmids.

XL-1 Blue E. coli (Novagen, USA) transformed specified a modified plasmid. Determined the sequence of bases of DNA isolated from transformed colonies, and thus received the plasmid pT14OSSI-2a-4T and pT14OSSI-2a-4T22Q containing a modified Shine-dalgarno sequence of the signal sequence enterotoxin.

In Fig.4 illustrates the above procedure for constructing vector pT14OSSI-2a-4T22Q respectively with obtaining transformants, designated NM 10603 and NM 10611 E. coli, which were deposited in the Korean culture collection of microorganisms (xsm) December 23, 1999, under access numbers xsm-10175 and xsm-10176, respectively.

In addition, vectors pT14OSSI-2b-4T and pT14OSSI-2b-4T22Q received in accordance with the procedure described above for vector pT14SSI-2b, which were used to transform BL21(DE3) E. coli with obtaining transformants, designated NM 10703 and NM 10711 E. coli, respectively. Transformants NM 10703 and NM 10711 E. coli were deposited in xsm 23 December 1999 under access numbers xsm-10177 and xsm-10178 respectively.

(3) Construction of a vector containing [Thr4,Val20, Gln22]STII

For replacing a 20 amino acid signal peptide sequence enterotoxin with Thr and Gln in the 4th and 22nd positions on the Val codon, vectors pT14OSSI-2a-4T22Q and pT14OSSI-2b-4T22Q obtained at stage (2), were subjected to PCR using oligonucleotides SEQ ID No: 29 and 30 by the method of site-directed mutagenesis described in stage (2), resulting in a received desired modified plasmid was obovale these modified plasmid. Determined base sequence, DNA isolated from transformed colonies, and thus received the plasmid pT14OSSI-2a-4T20V22Q and pT14OSSI-2b-4T20V22Q, which contained a gene with codons Thr, Val and Gln instead of Asp codons in the 4th position. Asp in the 20th position and Tyr in the 22nd position, respectively. BL21(DE3) E. coli transformed these plasmids with obtaining transformants, designated NM 10612 and NM 10712 E. coli, respectively.

Example 2. Obtaining mutant sequences Shine-Dalgarno for thermostable enterotoxin II

To reduce the number of bases between the binding site with the ribosome and initiating colon ATG modified signal sequences of thermostable enterotoxin II of E. coli within the sequence of the Shine-Dalgarno thermostable enterotoxin II obtained as described above, expressing the vector designed a modified plasmid in accordance with the procedure site-directed mutagenesis described in comparative example 2.

Namely to reduce the number of bases between the binding site with the ribosome GAGG and initiating ATG codon from 7 to 5 vector pT14OSSI-2a-4T22Q obtained, a description of the receiving oligonucleotides SEQ ID No: 31 and 32, as a result, we received a modified plasmid, designated pT14NSSI-2a-4T22Q. In addition, to reduce the number of bases between the binding site with the ribosome GAGG and initiating codon ATG to 4, the vector pT14NSSI-2a-4T22Q was subjected to the procedure site-directed mutagenesis described in comparative example 2 using the oligonucleotides SEQ ID No: 33 and 34, resulting in the received modified plasmid, designated pT14MSSI-2a-4T22Q.

XL-1 Blue E. coli transformed these modified plasmid. Determined base sequence, DNA isolated from transformed colonies, and thus received IFN-expressing plasmids pT14NSSI-2a-4T22Q and pT14MSSI-2a-4T22Q, which respectively contain 5 and 4 of the base between the binding site with the ribosome GAGG and initiating codon ATG. BL21(D3) E. coli transformed specified expressing plasmids with obtaining transformants, designated NM 10613 and NM 10614 E. coli, respectively.

Example 3. Comparison of the levels of expression of IFN-2

Transformants obtained as described above in Comparative ¡of these cultures were centrifuged at 6000 rpm for 20 minutes to precipitate bacterial cells, and the precipitate was processed by osmotic shock (Nossal, G. N., J. Biol.Chem., 241:3055, 1966), as described below.

The precipitate suspended in 1/10 volume of isotonic solution (20% sucrose, 10 mm buffer Tris-Cl containing 1 mm EDTA, pH 7.0). The resulting suspension was left for 30 minutes at room temperature, and then centrifuged to collect bacterial cells. The obtained cells resuspendable in D. W. when 4For extraction of proteins present in periplasm cells, and centrifuged to obtain the supernatant as periplasmatic solution. The level of IFN-2 in periplasmatic solution was analyzed using ELISA method (Kato, K. et al., J. Immunol., 116, 1554, 1976) using antibodies against IFN-2 (R&D, USA), and this level was calculated as the number of IFN-2a produced per 1 l of culture. The results are presented in the table.

Example 4. Post-treatment and purification

In accordance with the procedure described in example 3, the transformant NM 10611 E. coli, obtained as described in example 1(2), were cultured in LB medium and the culture was centrifuged at 6000 rpm for 20 minutes to collect cells. From these cells and sang and danced to pH 5.0-5.5, was adsorbing to a column of S-separate (Pharmacia Inc., Sweden), previously equilibrated to a pH of 5.3, and then the column is washed with 25 mm NaCl. IFN-2 was suirable by successive addition of the solutions, sauverny acetic acid containing 50 mm, 100 mm, 200 mm and 1 M NaCl, respectively, and the fractions containing IFN-2, collected and combined.

Combined fractions were subjected to chromatography on a column of separate Blue (Pharmacia Inc., Sweden) and suirable added in column buffer solutions containing more than 2 M NaCl, to obtain the active fraction.

The active fraction were dialyzed buffer, and finally subjected to fractionation on a column of anion exchange resin DEAE at pH of 5.8 with receiving IFN-2a, having a purity of more than 99%. In addition, IFN-2b was isolated from transformant NM 10711 E. coli by repeating the above procedure.

Each of the purified fractions IFN-2a and IEN-2b were subjected to electrophoresis in polyacrylamide gel with sodium dodecyl sulfate (LTOs-page) to determine purity and approximate concentrations of IFNand then subjected to the standard ELISA method as described is moreover, through analysis of N-terminal amino acid sequence was confirmed that IFN-2a and IFN-2b are natural types that do not contain additional methionine.

Example 5: Determination of molecular weight of IFN-2a produced from recombinant cell lines

The level of expression and the molecular weight of IFN-2a and IFN-2b produced from recombinant cell lines was determined by electrophoresis in LTO-page and Western blotting.

First periplasmatic fraction transformant NM 10611 E. coli, obtained as described in example 4 and purified IFN-2a, derived from it, were subjected to electrophoresis in LTO-page standard method using commercially available product IFN-2a (3106IU/ml) as control.

In Fig.5A shows the results of electrophoresis in SDS page with LTOs, where lane 1 corresponds to IFN-2A-control; lane 2 corresponds to periplasmatic fraction transformant NM 10611 E. coli; lane 3 corresponds to the purified IFN-2a, and is present in periplasmatic fraction transformant NM 10611 E. coli at a high level.

In addition, periplasmatic fraction transformant NM 10711 E. coli, purified fraction obtained by chromatography periplasmatic solution on a column of S-Separate, and finally, purified IFN-2b were subjected to electrophoresis in SDS page with the LTO standard method.

Nitrocellulose filter (Bio-Rad Lab, USA) was dipped in the buffer solution for blotting (170 mm glycine, 25 mm TrisHCl [pH 8], 20% methanol) and separated proteins on the gel were transferred to nitrocellulose filter for 3 hours using a set for blotting. The filter was kept in 1% casein for 1 hour, and three times washed with PBS containing 0.05% Tween-20. The filter is then placed in a solution of rabbit antibodies against IFN(Chemicon, # AB1434, USA), diluted PBS, and left for 2 hours at room temperature to complete the reaction. After the reaction, the filter 3 times washed with PBST solution to remove unreacted antibody. Then added conjugated with horseradish peroxidase goat antibody against rabbit IgG (Bio-Rad Lab, USA), diluted in PBS, and subjected to reaction at komnatnaya (Bio-Rad Lab, USA) for the development of the color reaction. The results of the above Western blot are shown in Fig.5b, where lane 1 corresponds to periplasmatic fraction transformant NM 10711 E. coli; lane 2 corresponds to the fraction purified by chromatography on a column of S-separate; and lane 3 corresponds to the final purified IFN-2b.

The results of this example confirm that the recombinant strains of E. coli of the present invention is expressed large amounts of soluble IFN.

Claims

1. Expressing the vector for the secretory production of human interferon alpha (hIFNin bacterial cells containing polynucleotide encoding a modified signal sequence of thermostable enterotoxin II, obtained by replacing one or more of the 4-th, 20-th and 22nd amino acids of the signal sequence of thermostable enterotoxin II E. coli having the amino acid sequence of SEQ ID No:3, amino acid selected from the group consisting of threonine, glutamine and valine; and polynucleotide encoding hIFN, legirovannye with his 3

3. Expressing the vector in p. 1, characterized in that the specified polynucleotide encoding hIFNencodes IFN-2a SEQ ID No:l or IFN-2b SEQ ID No: 2.

4. Expressing the vector for the secretory production of human interferon alpha (hIFNin bacterial cells containing polynucleotide encoding a modified signal sequence of thermostable enterotoxin II, obtained by replacing one or more of the 4-th, 20-th and 22nd amino acids of the signal sequence of thermostable enteada of threonine, glutamine and valine; polynucleotide encoding hIFN, legirovannye with his 3by the end, and the Shine-dalgarno sequence thermostable enterotoxin II E. coli (SD sequence, SEQ ID No: 8) or its mutant, legirovannye before 5-end of polynucleotide encoding a modified signal sequence of thermostable enterotoxin II.

5. Expressing the vector in p. 4, characterized in that the mutant SD sequence is a sequence obtained by deletion of 1 or 2 nucleotides from the area behind the GAGG, located on 5-end of SEQ ID No:8.

6. Expressing the vector in p. 4, characterized in that the mutant SD sequence has the nucleotide sequence SEQ ID No:9.

7. Expressing the vector in p. 1, characterized in that it is chosen from the group consisting of plasmids pT14SSI-2a-4T, pT14OSSI-2a-4T, pT14SSI-2a-4T22Q, pT14OSSI-2a-4T22Q, pT14OSSI-2a-4T20V22Q, pT14NSSI-2a-4T22Q, pT14MSSI-2a-4T22Q, pT14SSI-2b-4T, pT14OSSI-2b-4T, pT14OSSI-2a-4T (NM 10603, the access number xsm-10175) for expression of human interferon alpha.

9. The Escherichia coli strain BL21(DE3)/pT14OSSI-2a-4T22Q (NM 10611, the access number xsm-10176) for expression of human interferon alpha.

10. The Escherichia coli strain BL21(DE3)/pT14OSSI-2b-4T (NM 10703; the access number xsm-10177) for expression of human interferon alpha.

11. The Escherichia coli strain BL21(DE3)/pT14OSSI-2b-4T22Q (NM 10711; the access number xsm-10178) for expression of human interferon alpha.

12. The way secretory production of hIFNno additional methioninamide residue attached at its N-Terminus, where the method includes transforming bacterial cells expressing vector for secretory production of hIFNunder item 1 and cultivate these transformed bacterial cells.

13. The way secretory production of hIFNno additional methioninamide residue attached at its N-Terminus, where the method includes transforming bacterial cells expressing vector for secretory production of hIFN

 

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