Background of the Invention Isaacs and Lindenmann reported in 1957 that when chicken is infected with influenza virus A, a viral replication inhibitory factor designated interferon is produced (Isaacs, K and Lindenmann, J. Proc. R.

Soc. Lond., B147 : 258-267, 1957).

Human interferons are cytokine proteins which inhibit in vivo immune response or viral replication and they are classified as interferon alpha (IFNa), interferon beta (IFNO) and interferon gamma (IFN) according to cell types producing them (Kirchner, H. et al., Tex. Rep. Biol. Med., 41: 89- 93,1981; Stanton, G. J. et al., Tex. Rep. Biol. Med., 41: 84-88,1981).

It is well-known that these interferons work together to exert synergic effects in the manifestation of anti-viral, anti-cancer, NK (natural killer) cell activation and marrow cell inhibition activities (Klimpel, et al., J.

Imrnunol., 129: 76-78,1982; Fleischmann, W. R. et al., J Natl. Cancer Inst., 65: 863-966,1980; Weigent, et al., Infec. Immun., 40: 35-38,1980). In addition, interferons act as regulatory factors of the expression, structure and function of genes in the cell, and show a direct anti-proliferating effect.

IFNa is produced when leukocyte is stimulated by B cell mitogen, virus or cancer cells. Up to now, there have been reported genes that encode more than 20 species of interferons, each comprising 165 or 166 amino acids.

IFNa used for early clinical tests were obtained from buffy coat leukocyte stimulated by Sendai virus and its purity was only less than 1% (Cantell, K. and Hirvonen, Tex. Rep. Biol. Med., 35 : 138-144, 1977).

It has become possible to produce a large quantity of IFNa having biophysical activity by gene recombinant techniques in the 1980' (Goedell, D. V et al., Nature, 287: 411-416,1980). Clinical tests using the recombinant hIFNa have shown that it is effective in treating various solid cancers, particularly bladder cancer, kidney cancer, HIV related Kaposi's sarcoma, etc. (Torti, F. M., J Cli. Oz2col., 6: 476-483, 1988 ; Vugrin, D., et al., Cancer Treat. Rep., 69 : 817-820,1985; Rios, A., et al., J Ctin. 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 its applicable range as a therapeutic agent is expanding day by day.

The result of cloning IFNa gene from leukocyte has shown that IFNa is encoded by a group of at least 10 different genes. This indicates that the DNA sequences of the genes do not produce one kind of protein but that IFNa is a mixture of subtype proteins having similar structures. Such subtype proteins are named IFNa-1, 2,3, and so on (Nature, 290: 20-26, 1981).

Among the several types of interferons, hIFNa purified from human leukocyte has a molecular weight of 17,500 to 21,000, and a very high native activity of about 2 X 108 lU/mg protein. In vivo IFNa is a protein consisting of 165 amino acids. It is designated IFNa-2a (SEQ ID NO: 1) in case the 23rd amino acid is lycine, and IFNa-2b (SEQ ID NO: 2) in case the 23rd amino acid is arginine. In the beginning, hIFNa was produced by a process using a cell culture method. However, this process is unsuitable for commercialization because of its low productivity of about 250 ug/L.

To solve this problem, processes for recovering a large quantity of

interferon from microorganisms by using gene recombinant techniques have been developed and used to date.

The most widely employed is a process using E. coli which produces IFNa consisting of 166 or 167 amino acids according to the characteristics of the E. coli cell. These products have an extra methionine residue added at the N-terminal by the action of the ATG codon existing at the site of initiation codon. However, it has been reported that the additional methionine residue can trigger harmful immune response, in the case of human growth hormone (EP Patent Publication No. 256,843).

In addition, most of the expressed IFNa accumulates in cytoplasm in the form of insoluble inclusion bodies and must be converted into an active form through refolding during a purification process. As such a refolding process is not efficient, IFNa exists partially in a reduced form, or forms an intermolecular disulfide coupling body or a defective disulfide coupling body.

It is difficult to remove these by-products, which cause a markedly low yield.

In particular, it is extremely difficult to remove undesirable interferon by- products such as misfolded interferons.

Recently, in order to solve the above mentioned problems associated with the production of a foreign protein within a microbial cell, various efforts have been made to develop a method based on efficient secretion of a soluble form of the target protein carrying no extra methionine added to the N-terminal.

In this method, a desired protein is expressed in the form of a fusion protein which carries a signal peptide attached to its N-terminal. When the fusion protein passes through the cell membrane, the signal peptide is removed by an enzyme in Ecoli and the desired protein is secreted in a native form.

The secretive production method is more advantageous than the microbial production method in that the amino acid sequence and the higher structure of the produced protein are usually identical to those of the wild- type. However, the yield of a secretive production method is often quite low due to its unsatisfactory efficiencies in both the membrane transport and

the subsequent purification process. This is in line with the well-known fact that the yield of a mammalian protein produced in a secretory mode in prokaryotes is much lower than that of a prokaryotic protein produced in the same mode in prokaryotes. Therefore, it has been attempted to develop a more efficient secretory production method. For instance, Korean Patent Publication No. 93-1387 discloses an attempt to mass-produce IFNa using the signal peptide of E. coli alkaline phosphatase, but the yield was very low at 109IU/L culture medium (10 ug/L culture medium). Therefore, there has been a keen interest in developing a method which is capable of producing soluble IFNa having no additional methionine residue added at the N- terminal, using a microorganism on a large scale.

The present inventors have previously generated a new signal peptide of E. coli thermostable enterotoxin II (Korean Patent Application No.

98-38061 and 99-27418) and found that this new secretory signal peptide can be used for the mass-production of the native form of IFNa. Namely, the present inventors have constructed an expression vector containing a gene obtained by ligating IFNa encoding gene instead of enterotoxin II encoding gene to the modified E. coli secretory signal peptide, and developed a secretory production method of IFNa having a native biological activity via the microbial secretory system by culturing the microorganism transformed with said expression vector.

Summary of the Invention Accordingly, it is an object of the present invention to provide an expression vector which can secretively produce human interferon alpha (mina).

It is another object of the present invention to provide a microorganism transformed with said expression vector.

It is a further object of the present invention to provide a process for producing a soluble form of hIFNa using said microorganism, which has no extra methionine residue attached to the amino terminus.

Brief Description of the Drawings The above and other objects and features of the present invention will become apparent from the following description of the invention taken in conjunction with the following accompanying drawings; which respectivelyshow: Fig. 1 : the procedure for constructing vector pT-IFNa-2a ; Fig. 2: the procedure for constructing vector pTl4SIa-2a ; Fig. 3: the procedure for constructing vector pT14SSIa-2a ; Fig. 4: the procedure for constructing vector pT140SSIa-2a-4T22Q ; Figs. 5a and 5b : the results of SDS-PAGE which verify the expression of IFNa-2a and the purity of the expressed IFNa-2a from recombinant cell lines, and the result of western blot analysis which verifies the molecular weight of expressed IFNa-2b, respectively.

Detailed Description of the Invention According to one aspect of the present invention, there is provided an expression vector for the secretive production of hIFNa comprising a polynucleotide encoding a modified thermostable enterotoxin II signal sequence (hereinafter, as referred as to'STII mutant') and a polynucleotide encoding hIFNa ligated to the 3'-end thereof.

The polynucleotide encoding hIFNa used for constructing the expression vector of the present invention may be any one of polynucleotides encoding random hIFNa subtypes such as native hIFNa-2a (SEQ ID NO: 1), IFNa-2b (SEQ ID NO: 2), IFNa-I and IFNa-3, and it may also be a recombinant polynucleotide which has a modified base sequence that encodes any of the above IFNa subtypes.

The polynucleotide encoding the modified E. coli thermostable enterotoxin II signal sequence of the present invention, which is ligated to

the front of the 5'-end of the polynucleotide encoding hIFNa and used for the purpose of the secretive production of hIFNa, may be a polynucleotide encoding a mutant derivable by replacing one or more of the amino acids of E.coli thermostable enterotoxin II signal sequence described in SEQ ID NO : 3, preferably one or more of the 4 «, 20 and 22nd amino acids thereof with other amino acid (s). Examples of such polynucleotides encode mutants obtained by replacing: the 4 amino acid with threonine ( [Thr4] STTI) ; the 4th amino acid with threonine and the 22nd amino acid with glutamine, respectively ([Thr4, Gln22] STII) 7 the 4 amino acid with threonine, the 20 amino acid with valine and the 22nd amino acid with glutamine, respectively ([Thr4, Val20, Gln22] STII), and the 4th amino acid with threonine and the 20 amino acid with valine, respectively ([Thr4, Val20] STII) in the E. coli thermostable enterotoxin II signal sequence (STII) described in the SEQ ID NO: 3, and preferred polynucleotide sequences are SEQ ID NOS: 4,5,6 and 7. However, it is known that several different polynucleotides encoding the mutants of the present invention may exist due to the codon degeneracy, and, specifically, a polynucleotide modified by introducing preferred codons of E.coli without any change of amino acid sequence can be used for promoting the expression rate ofIFNa.

In addition, the expression vector of the present invention may further comprise E.coli thermostable enterotoxin II Shine-Dalgarno sequence (SD sequence, SEQ ID NO: 8) or its mutant ligated to the front of the 5'-end of the polynucleotide encoding the modified thermostable enterotoxin II signal sequence. As compared with an wild-type which has 7 bases (TGATTTT) following GAGG of the 5'-end in the E.coli thermostable enterotoxin II SD sequence described in the SEQ ID NO: 8, the mutant of SD sequence has a shorter sequence of 6 or 5 bases. The use of this mutant can increase the secretive expression rate of IFNot. However, when said base sequence becomes shorter than 4 bases, the expression rate decreases markedly. A specific example of a preferred mutant that can be used in the present invention is the E. coli thermostable enterotoxin II SD sequence mutant having the nucleotide sequence of SEQ ID NO: 9.

The promoter used in preparing the expression vector of the present invention may be any of those which can express a heterologous protein in a microorganism host. Specifically, lac, Tac, and arabinose promoter is preferred when the heterologous protein is expressed in E. coli.

This invention also provides transformed microorganisms which may be obtained e. g., by transforming such E. col strains as E. coli BL21 (DE3) (Novagen, USA) or E. coli XL-1 blue (Novagen, USA) with said expression vector. Examples of the present invention provide such transformed microorganisms: E. coli BL21 (DE3)/pT140SSIa-2a-4T ("HM 10603"), E. coli BL21 (DE3)/pT140SSIa-2a-4T22Q ("HM 10611"), E. coli BL21 (DE3)/pT140SSIoc-2b-4T ("HM 10703") and E. coli BL21 (DE3)/pT140SSIa-2b-4T22Q ("HM 10711"). The above transformed microorganisms are deposited in Korean Culture Center of Microorganisms (KCCM) (Address; Yurim Bldg., 361-221, Hongje 1-dong, Seodaemun-gu, Seoul 120-091, Republic of Korea) on December 23,1999 under accession numbers KCCM-10175, KCCM-10176, KCCM-10177 and KCCM-10178, respectively, in accordance with the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganism for the Purpose of Patent Procedure.

In accordance with another aspect of this invention, there is also provided a process for secretively producing hIFNa having no additional methionine residue attached at the N-terminal, into the periplasm of Ecoli by culturing the transformed microorganism under an appropriate culture condition which may be the same as the conventional culture condition used for transformed microorganisms. hIFNa secretively produced by the process of the present invention comprises random hIFNa subtypes such as IFNa-1, IFNa-3 and so on, as well as native hIFNa-2a (SEQ ID NO: 1) and hIFNa-2b (SEQ ID NO: 2) consisting of 165 amino acids. In addition, the process of the present invention can be applied to the production of any other interferon such as hIFNß and hIFNy.

According to the process of the present invention, 80% or more of

IFNa produced by the inventive E. coli transformant is secreted into the periplasm at a high productivity of more than 1 g/L. The produced IFNa has the same amino acid sequence as that of native IFNa which has no additional amino acid attached at the N-terminal, and shows a biological activity equal to that of native IFNa.

The following Examples are included to further illustrate the present invention without limiting its scope.

Reference Example : IFNa-2a gene and construction of a vector containing same A gene encoding hIFNa-2a was prepared by carrying out PCR using human genomic DNA as a template and SEQ ID NOS: 10 and 11 as primers.

The primer of SEQ ID NO: 10 was designed to provide an NdeI restriction site (5'-CATATG-3') upstream from the codon for the first amino acid (cysteine) codon of native hIFNa, and the primer of SEQ ID NO: 11, to provide a BamHI restrction site (5'-GGATCC-3') downstream from the termination codon thereof.

The amplified PCR product was cleaved with NdeI and BamHI to obtain a DNA fragment encoding hIFNa-2a. The DNA fragment was inserted into the NdeI/BamHI site of vector pET-14b (Novagen, USA) to obtain vector pT-IFNa-2a.

Fig. 1 shows the above procedure for constructing vector pT-IFNa- 2a.

Comparative Example 1: Construction of a vector containin enterotoxin signal sequence and IFNa-2a genes To prepare E. coli enterotoxin II signal sequence gene, the pair of complementary oligonucleotides of SEQ ID NOS: 12 and 13 were designed based on the previously known nucleotide sequence of E. coli enterotoxin II

signal peptide, and synthesized using a DNA synthesizer (Model 380B, Applied Biosystem, USA). The above oligonucleotides were designed to provide a BspHI restriction site (complementary sites to an Ndel restriction site) upstream from the initiation codon of E. coli enterotoxin II and a Mlul restriction site introduced by a silent change at the other end. Both oligonucleotides were annealed at 95°C to obtain a blunt-ended DNA fragment having a nucleotide sequence encoding Ecoli enterotoxin II signal sequence. The above DNA fragment was inserted into the SmaI site of vector pUCl9 (BioLabs, USA) to obtain vector pUC19ST.

In addition, vector pT-IFNa-2a containing IFNa-2a gene obtained in Reference Example was subjected to PCR using the primers of SEQ ID NOS: 14 and 15 to ligate the enterotoxin signal peptide to IFNa-2a gene.

The primer of SEQ ID NO: 14 was designed to correspond to the 5'-end of IFNa-2a gene, and the primer of SEQ ID NO: 15, to provide a BamHI restriction site (5'-GGATCC-3') downstream from the termination codon thereof. The DNA fragment containing the polynucleotide, which encodes native IFNa-2a, was amplified by PCR using the above polynucleotide primers. The amplified DNA fragment was cleaved with Mlul and BamHI to obtain an IFNa-2a DNA fragment having MluI/BamHI ends.

Meanwhile, vector pUC19ST containing the enterotoxin signal peptide was cleaved with Mlul and then digested with BamHI to obtain a vector fragment having MluI/BamHI ends. The vector fragment was ligated to the IFNa-2a DNA fragment to construct vector pUC 19SIFNa-2a.

Vector pUC19SIFNa-2a was cleaved with BspHI and BamHI to obtain a DNA fragment (564 bp). The DNA fragment was inserted at the NcoI/BamHI section of vector pET-14b (Novagen, USA) to obtain vector pT14SIa-2a. Fig. 2 shows the above procedure for constructing vector pTl4SIoc-2a.

Subsequently, E. coli BL21 (DE3) strain was treated with 70 mM calcium chloride solution to prepare competent E. coli, and then, vector pT14Ia-2a in 10 mM Tris buffer (pH 7.5) was added thereto. An E. coli transformant expressing IFNa-2a was selected by a conventional method

which exploits the sensitivity of the transformed vector toward antibiotics, and designated E. coli HM 10600.

In addition, vector pT14SIa-2a was subjected to PCR using the primers of SEQ ID NOS: 16 and 17 to amplify a DNA fragment obtained by ligating the Shine-Dalgarno sequence of the enterotoxin, the enterotoxin signal peptide, and IFNa-2a gene, in that order, and then the DNA fragment was cleaved with Xbal and BamHI to obtain an insert.

The insert was ligated into the XbaI/BamHI section of vector pET- 14b (Novagen, USA) to construct vector pT14SSIa-2a. Fig. 3 displays the above procedure for constructing vector pT13SSIa-2a. E. coli BL21 (DE3) (Stratagene, USA) was transformed with vector pT14SSIa-2a to obtain a transformant designated E.coli HM 10601.

Comparative Example 2: Construction of a vector containing enterotoxin signal sequence and IFNa-2b genes The 23rd lycine codon of IFNa-2a gene in vector pT14SSIa-2a was replaced by arginine codon with a site-directed mutagenesis (Papworth, C. et al., Stratagies, 9,3,1996) to construct an expression vector containing IFNa-2b gene. Vector pT14SSIa-2a was subjected to hybridization with the synthetic oligonucleotides of SEQ ID NOS: 19 and 20 containing the replaced codon to form a hybrid molecule and DNA amplification was performed using pfu (Stratagene, USA) and four nucleotide triphosphates (ATP, GTP, TTP, CTP) which extend said oligonucleotides in the 5'-3' direction.

Interferon a-2b sequence

The amplified DNA fragment was recovered and an restriction enzyme Dpnl was added thereto to remove unconverted plasmids completely.

E. coli XL-1 blue (Novagen, USA) was transformed with the modified plasmid. The base sequence of the DNA recovered from transformed colonies was determined, and thus obtained was plasmid pT14SSIa-2b which contained a gene having arginine in place of the 23rd amino acid lycine of IFNa-2a.

Subsequently, E. coli BL21 (DE3) was transformed with the modified vector pT14SSIa-2b to obtain a transformant designated E. coli HM10701 by using the same method described in Comparative Example 1. By analyzing the N-terminal amino acid sequence of the protein produced by culturing the transformant, it has been confirmed that IFNa-2b having the native amino acid sequence was expressed therefrom.

Example 1 : Construction of a vector containing enterotoxin signal peptide mutant (1) Construction of a vector containing [Thr41STII In order to modify a specific amino acid residue of the enterotoxin signal sequence peptide, a vector containing a polynucleotide encoding enterotoxin mutant signal sequence was prepared by site-directed mutagenesis as follows.

First, vector pT14SSIa-2a obtained in Comparative Example 1 was subjected to PCR using oligonucleotides of SEQ ID NOS: 22 and 23 to obtain a modified plamid, wherein the 4 amino acid of the enterotoxin signal sequence is replaced with threonine (Thr), by the site-directed mutagenesis procedure described in Comparative Example 2.

Then, E.coli XL-1 blue (Novagen, USA) was transformed with the modified plasmid. The base sequence of DNA recovered from the transformed colonies was determined, and thus obtained was a plasmid which contained a gene encoding the enterotoxin signal sequence peptide having Thr in the 4 amino acid position thereof. The plasmid thus obtained was cleaved with XbaI and MM, and then inserted into the Xbal/MluI section of vector pT14SSIa-2a to obtain vector pT14SSIa-2a-4T.

Subsequently, E. coli BL21 (DE3) (Stratagene, USA) was transformed with vector pT14SSIa-2a-4T to obtain an E. coli transformant designated E.coZiHM 10602.

Vector pT14SSIa-2a-4T was constructed using pT14SSIa-2b, and then transformed into Ecoli BL21 (DE3) (Stratagene, USA) to obtain an E. coli transformant designated E. coli HM 10702 by the same method described above.

(2) Construction of a vector containing [Thr4, Gln221 STII Vector pT14SSIa-2a-4T obtained in step (1) was subjected to PCR using the oligonucleotides of SEQ ID NOS: 25 and 26, which were designed to substitute Gln codon for the 22nd amino acid of the enterotoxin signal peptide having Thr in its 4al position, in accordance with the site-directed mutagenesis procedure of step (1) to obtain a modified plasmid.

Then, E.coli XL-1 blue (Novagen, USA) was transformed with the modified plasmid. The base sequence of DNA recovered from transformed colonies was determined, and thus obtained was plasmid pT14SSIa-2a- 4T22Q which contained a gene having Thr and Gln in the 4th and 22nd amino

acid positions of the enterotoxin signal sequence, respectively.

Subsequently, E. coli BL21 (DE3) (Stratagen, USA) was transformed with vector pT14SSIa-2a-4T22Q by the same method described in step (1) to obtain a transformant designated E. coli HM 10604.

To modify the Shine-Dalgarno sequence of the modified enterotoxin signal sequence into SEQ ID NO: 9, vectors pT14SSIa-2a-4T and pT14SSIa-2a-4T22Q were subjected to the site-directed mutagenesis procedure described in step (2) using the oligonucleotides of SEQ ID NOS : 27 and 28 to obtain the desired modified plasmid.

E. coli XL-1 blue (Novagen, USA) was transformed with the modified plasmid. The base sequence of the DNA recovered from transformed colonies was determined, and thus obtained were plasmids pT140SSIa-2a-4T and pT140SSIa-2a-4T22Q having modified Shine- Dalgarno sequence of enterotoxin signal sequence. Fig. 4 represents the above procedure for constructing vector pT140SSIa-2a-4T22Q.

E. coli BL21 (DE3) was transformed with vector pT140SSIa-2a-4T and pT140SSIa-2a-4T22Q, respectively, to obtain a transformant designated E. coli HM 10603 and HM 10611, which were deposited in Korean Culture Collection of Microorganisms (KCCM) on December 23,1999 under accession numbers KCCM-10175 and KCCM-10176, respectively.

In addition, vectors pT140SSIa-2b-4T and pT140SSIa-2b-4T22Q were prepared by the same procedure as above using vector pT14SSIa-2b, which were used to transform E. coli BL21 (DE3) to obtain transformants designated E. coli HM 10703 and HM 10711, respectively. E. coli transformants HM 10703 and HM 10711 were deposited in KCCM on December 23,1999 under accession numbers KCCM-10177 and KCCM- 10178, respectively.

(3) Construction of a vector containing [Thr4, Val20, Gln22] STII To further substitute Val codon for the 20th amino acid of the enterotoxin signal sequence peptide having Thr and Gln in its 4 and 22nd

amino acid positions, vectors pT140SSIa-2a-4T22Q and pT140SSIa-2b- 4T22Q prepared in step (2) were subjected to PCR using the oligonucleotides of SEQ ID NOS: 29 and 30 by the site-directed mutagenesis procedure described in step (2), to obtain the desired modified plasmids designated pT140SSIa-2a-4T20V22Q and pT140SSIa-2b- 4T20V22Q.

E. coli XL-1 blue was transformed with the modified plasmids. The base sequences of the DNAs recovered from transformed colonies were determined, and thus obtained were plasmids pT140SSIa-2a-4T20V22Q and pT140SSIa-2b-4T20V22Q which contained a gene having Thr, Val and Gln codons in places of the 4 Asp, 20th Asp and 22nd Tyr codons, respectively. E. coli BL21 (DE3) was transformed with the plasmids to obtain thransformants designated E. coli HM 10612 and HM 10712, respectively.

Example 2: Preparation of thermosrable enterotoxin II Shine-Dalwarno sequence mutant In order to reduce the number of bases between the ribosome binding site and initiation codon ATG of the modified E. coli thermostable enterotoxin II signal sequence within thermostable enterotoxin II Shine- Dalgarno sequence of the above-prepared expression vector, a modified plasmid was constructed by the site-directed mutagenesis procedure of Comparative Example 2.

Namely, to reduce the number of bases between the ribosome binding site GAGG and initiation codon ATG from 7 to 5, vector pT140SSIa-2a-4T22Q prepared in Example 1 (2) was subjected to the site- directed mutagenesis procedure of Comparative Example 2 using the oligonucleotides of SEQ ID NOS: 31 and 32 to obtain a modified plasmid designated pT14NSSIoc-2a-4T22Q. In addition, to reduce the number of bases between the ribosome binding site GAGG and initiation codon ATG to 4, vector pT14NSSIa-2a-4T22Q was subjected to by the site-directed

mutagenesis procedure of Comparative Example 2 using the oligonucleotides of SEQ ID NOS: 33 and 34 to obtain a modified plasmid designated pT14MSSIa-2a-4T22Q.

E. coli XL-1 blue was transformed with the modified plasmids.

The base sequences of the DNAs recovered from transformed colonies were determined, and thus obtained were IFNa expression plasmids pT14NSSIa- 2a-4T22Q and pT14MSSIa-2a-4T22Q which respectively contained 5 and 4 bases between the ribosome binding site GAGG and initiation codon ATG.

E. coli BL21 (DE3) was transformed with the expression plasmids to obtain transformants designated HM 10613 and HM 10614, respectively.

Example 3: Comparision of expression amount of IFNa-2 Transformants prepared in the above Comparative Examples and Examples were cultured in LB medium and then incubated in the presence of IPTG for 3 hours. Each of the cultures was centrifuged at 6,000 rpm for 20 min. to precipitate bacterial cells and the precipitate was treated by Osmotic shock method (Nossal, G. N., J. Biol. Chem., 241: 3055,1966) as following.

The precipitate was suspended in a 1/10 volume of isotonic solution (20% sucrose, 10 mM Tris-Cl buffer containing 1 mM EDTA, pH 7.0). The suspension was allowed to stand at room temperature for 30 min, and then centrifuged to collect bacterial cells. The cells were resuspended in D. W. at 4°C to extract the proteins present in the periplasm of the cells, and centrifuged to obtain a supernatant as a periplasmic solution. The IFNa-2 level in the periplasmic solution was assayed in accordance with ELISA method (Kato, K. et al., J Immunol., 116, 1554,1976) using an antibody against the IFNa-2 (R&D, USA), which was calculated as the amount of the IFNa-2a produced per 1 Q of culture. The results are shown in Table 1.

Table I: Comparision of expression amount of IFNa-2 Transformant Example Expression Modified IFNα-2

Vector amino acid Level residue in STII in periplasm* HM 10600 Comp. PT14SIa-2a 8240 Exam.1 HM 10601 Comp. PT14SSIa-2a 325 75 Exam. 1 HM 10701 Comp. PT14SSIa-2b 28890 Exam. 2 HM 10602 Example pT14SSIa-2a-4TThP550 120 1(1) HM 10603 Example pT14OSSIα-2a- Thr4 1,020~135 1 (2) 4T HM 10604 Example PT14SSIα-2a- Thr4, Gln22 680~105d 1 (2) 4T22Q HM 10611 Example pT14OSSIα-2a- Thr4, Gln22 1,220~120 1 (2) 4T22Q HM 10612 Example pT14OSSI3035-2a Thr4, Val2, 130180 1(3) 4T20V22Q Gln22 HM 10613 Example PT14NSSIα-2a- Thr4, Gln22 750~144 2 4T22Q HM 10614 Example pT14MSSIα-2a- Thr4, 420100 2 4T22Q HM 10702 Example pT14SS3035-2b-4T Thr4 370~90 1(1) HM 10703 Example pT14OSSIα-2b- Thr4 735~117 1 (2) 4T HM 10711 Example pT14OSSIα-2b- Thr4, Gln22 1,070~150 1 (2) 4T22Q HM 10712 Example pT14OSSIα-2b- Thr4, 820160 1(3)4T20V22Q Gln²² * IFNa mg/100 O. D6oonm/L culture solution Example 4: Post-treatment and purification According to the procedure of Example 3, transformant E. coli HM 10611 prepared in Example 1 (2) was cultured in LB medium and the culture was centrifuged for 6,000 rpm for 20 min. to harvest cells. The periplasmic solution was prepared from the cells by the Osmotic Shock method.

The periplasmic solution was adjusted to pH 5.0 to 5.5, adsorbed on

an S-Sepharose (Pharmacia Inc., Sweden) column pre-equilibrated to pH 5.3, and then, the column was washed with 25 mM NaCl. IFNa-2 was eluted by sequentially adding acetic acid buffer solutions containing 50 mM, 100 mM, 200 mM and 1 M NaCI, respectively, and fractions containing IFNa-2 were collected and combined.

The combined fractions were subjected to Blue Sepharose (Pharmacia Inc., Sweden) column chromatography and eluted by adding to the column buffer solutions containing more than 2 M NaCI to obtain an active fraction.

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

Each of the purified IFNa-2a and IFNa-2b fractions was subjected to sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) to determine the purity and approximate IFNa concentration, and then subjected to a conventional ELISA method as in Example 3 to determine the exact IFNa concentration in the periplasmic solution. In addition, it was confirmed by N-terminal amino acid sequence analysis that IFNa-2a and IFNa-2b were of the native types having no additional methionine.

Example 5: Determination of IFNa-2a molecular weisht produced from recombinant cell lines The expression and molecular weights of IFNa-2a and IFNa-2b produced from recombinant cell lines were determined by using SDS-PAGE and Western blotting.

First, the periplasmic fraction of transformant E. coli HM 10611 prepared in Example 4 and purified IFNa-2a obtained therefrom were subjected to SDS-PAGE using a commercial IFNa-2a product (3 X 106 IU/ml) as a control according to the conventional method. Fig. 5a

reproduces the SDS-PAGE result, wherein lane 1 shows the IFNa-2a control; lane 2, the periplasmic fraction of E. coli transformant HM 10611 ; and lane 3, the purified IFNa-2a. As can be seen from Fig. 5a, the purified IFNa-2a had the same molecular weight as that of the native IFNa-2a, and was present in the periplasmic fraction of transformant E. coli HM 10611 at a high level.

In addition, the periplasmic fraction of transformant E. coli HM 10711, a purified fraction obtained by subjecting the periplasmic solution to S-Sepharose column chromatography and the finally purified IFNa-2b were subjected to SDS-PAGE according to the conventional method.

A nitrocellulose filter (Bio-Rad Lab, USA) was wetted with a buffer solution for blotting (170 mM glicine, 25 mM Tris'HC1 [pH 8], 20% methanol) and the proteins separated on the gel were transferred onto the nitrocellulose filter over a period of 3 hours by using a blotting kit. The filter was kept in 1% Casein for 1 hour and washed three times with PBS containing 0.05% Tween 20. The filter was put in a rabbit anti-IFNa antibody (Chemicon, #AB1434, USA) solution diluted with PBS and reacted at room temperature for 2 hours. After reaction, the filter was washed 3 times with a PBST solution to remove reacted antibody. Horseradish peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad Lab., USA) diluted with PBS was added thereto and reacted at room temperature for 2 hour.

The filter was washed with PBST, and a peroxidase substrate kit (Bio-Rad Lab., USA) solution was added thereto to develop a color reaction. The results from the above western blotting are shown in Fig. Sb, wherein lane 1 represents the periplasmic fraction of transformant E. coli HM 10711; lane 2, the fraction purified with S-Sepharose column chromatography; and lane 3, the final purified IFNa-2b.

As a result of Example, it is confirmed that a large quantity of soluble IFNa is expressed from the recombinant E. coli strains of the present invention. BUDAPEST TREATY OM THE JhJTERNATJONAL RECOGNmON OF THE DEPOSE OF MJCROORGlSMS FOR THE PURPOSED OF P.'YTEMT PROCEDURE llXlTEPu T10NAL FORhl r To. Hanini Phaj-m. Co... Ltd. , G93 5 H3jeo-ri Paltan-rnyun Hsurt-Kun RECEIPT IN THE CASE OF AN ORDINAL Kyong'S'J'eio. issued pursuant to Ru) < ; 7. 'by the Republic of Korea tNTERMATiOAL OEPO. S ! TAEY AUTHORITY idp. ntiBed at the bottom of this page L-J I. IDENTTFICATJOh) OF THE MICROORGANISM Identi. ficadon reference B'iven by tf') e AccMS) oa rutmber gjen by the DEPOSITOR : INTERNATIONAL DEPOSITARY AIJTHORTrY' : HM10603KCCM-10175 n. SCIBNTtFIC DESCRtPTtON ANP/OR. PROPOSED TAXOOMCC DESIGNATION The T'croorgantSfn) dennSsd under I sbove'M'as sccoTfipanjed. by ! Li a scientific dcscripdon O a proposed taxo. nomic desig'natjc'n (Mt-k with a cross where appUcabt' :) in. RECEIPT/1ND ACCEPTANCE : Tins'iNtGm&tJonal Depositary Authoui accepts the m<croot'gBn) R : m idcnt. ied uada' ! abce, which was' received by it 0) ! Dec. 23. 1S99 (date. of the origiDa ! deposit)' IV. INTERNATtONAL DEPOSITARY AUTHORITY clame : Korean Culture Center of MroorRRistDS SjnaHj) rf' (s) of Persns (s) having the power to represent the Internaiian : Depositary Xddress : 36 2') Auihority of of authoukcct officiat (s)- : , Seodaemlln-gu X æ WJ$m. Sodaemvn-gu- ! SEOUL 120-091 Date : Dec. 29. 1999.'= Republic of Norej. T) i 1 Where Rule 6. 4 (d) applies, such date is the date on which l. he status of int== Dsitst-auiorit- was acquireBrl where a deposit made outside the Budapest Treaty after the acquisition of the atatue of nterDational depositary authority is con-verted into a deposit v<3er the Plldapest trcaty, such dat. e is tlt date on which the inicrocrsfusm ws received by tha internationat dpo. ? itaiy tjouky. BUDAPEST TREATY OH THE IfTERN. T ! 0 ! \L RECOGNmON OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE iDITEFU TOs r-) To.HMn :)') Phnn. Cc'., D. d. r To. Ii:; J77T] 1 T'harm. C: c,, LI. d. ". Va S U nL7-I''U n HEunp-Kun RECEIPT M'THE CASE OF AN ORfGiN/YL Kyongi-do, ! S ! m ? d pur-HuMt t. o Ru ! & 7. bv the Republic of KoreH ITBR !'-t'nO) AL DEPOSlTY ADTHORrr.-' iriectvined at the clottonz vf lllis p3. ge . I.' [DENT ! F] CAT ! ON OF THE MICRO-ORGANISM JdRBttBcatioa rEfErence. jyjvcn by the AcceasJoo nnmber c'h'fn by the at :'IIVTFlttlTIDlV. gT pFCJ5iTil. R r'tlL'T110'P. I7'Y : nMjoenKCCM-0176 0 Iì SCIENTIFIC DESCRfPTfOM AD/OR RROPO. ? ED TXOOMIC DESGMATION T)')) ; inicrbot'ganistn identtfted UDder 1 abcTe was ttccoimpanitd'by D a aclentlflc 2e-cripe ; om O a. proposed taxooom ! c d. esatjoD (Mart TAhcrt-Lpplicbole,) 111. P. ECEIPT. AND AccE ; PTl\elcE '*'"..; ; Tl iS l. ni. ernational Depositr) f/auf. houiir accepts the Wicroorganisn iclentifie lnder I i1170ff.. hiciu was ., reeeixrecl bY ii : on IZec.. 2 99 (date of tbe origix eP<ssit)' 1,1 1,, IATI lll,, IAI ! V. INTERNATIONAJ. DEPOSITARY AUTHORITY 7 Name : Koyean Cu ! ti ! re Center of MicroorBsmsma SignMure (s) of person (s) LaviaS e power to represent the Jnlernadona1 DeposJtaly filr, idress : 3CI-22l, Bruz BS 1'zdd. res : 361221, r11T7I11 73/ lharity of of t7vihoui. ed officiai s) Authortty cf of euthou'zed hfficot (' Horlgjrl-dong, ne Seodaemlm-gru 1 WMg SEOUL 120-091 193te : Vec. 29. 1999. x Republic of liores 9 3361 r ), 11 t i i It J llere Ru ! e 6. 4 (d) tplies, sulr 4ai : e is dte dste on which the status o inlcHSnSeposha) ? suthorxy wsa acqun'ed : Where a deposit made otsMc [he. Budapest TreaLy aAer the acquion of the a (a<as oJ') nto'nadona ! depos ; bry ahori is converted into a deposit unds'the Budapest Treats', such <e is the date M hich the DiicroorpMism was reccivRd by the internatiooa ? < ! cposft3r) r aud') ou')'b'. BUDAPEST TREATY ON THE ! NTERNAnONAL RECOGN'l'nON OF THE DEPOSIT OF FORTHE. FURPOS& ? OP FAMWT PROCEDURE TtiTED IAT tL 1 OIii\./f r To. H X nmi Pharßnv C. 893-5 Hujeo-ri Pairn-myun Hxl, asung-liun RFCCEIPT IX HE CASE CF hN ORIC71Ni Kyonpg'i-do. issued pursusnt to Rtile 7,) b3, Lbe Repub)) c of Korea ITERATiOM. AL DEPOSIT/RY AIJTHOR ! TV kien'ified at tiie box rJ of this puge L X I. IDENTIFICATION OF THE MICROORGANISM id hl-fL-, CL4t3i ol IdentiGcatioa r' :. ference give by the Accession number pEn. by the DEPOSlTOPu iNEN. ATIOS, D DEFOSIThRtY AWS HM10703KCCM-10177 D. SCIEMi ; FTC DESm-tON AND/OR PROPOSED T.-tONOMTC DT-t ; IGtl, NTlON The mlcroor. ganism identi£ied undc bove ma aScompßnled bY Cl a scienttrfir dcsc7iption Cl a proposed, tnxojtonuc des'gTiBHon (lferSs o : ith a crçss where aPpIiab7e) S] :. RECEIPT AMD ACCEfTAMCE : ThisInternationa.) Dcpositaf)'Authority aceep'ts the. nDcroorganjm idetiSed under I above, which'm. s recei-v&d by it on Dec. 23. 1999 (dat of the orinal deposit)' IV. IITTERNATION DEPOSITARY AUTHORITY Ma. ma : KoreaTt Cu ! ture Center ofMicroorfrajDisms 5} CMtut-e (s) of person (9) having the power to represent the Int¢rn2tional P¢posiiaxy Ad.rei) r' Authorlty uf Pf authouiged of ficial (s) : H°ngic-l-dong, = Seodaemun-gu e SEtO L 1 ? o-09l LUatc : Dec. 29. 1999. l RcpubJ. ic of Koica '**'') ''X'-' I'Where Ru) e 6. 4. (d) 3pp ! ies. such date is the date on which the status Of inc't.. r.'SJtflfY aUrJli) Clfy was acquired : N ere a deposit msde outside the Budapest Treaty <'s)'the a< : qttjs ? t'pn c d'"s status of' nternationat deposjfar ; authon is converted into a deposit under tre Budapest Treaty', su. ch date'$ the. date on sffiich the microorganism was received by tht intearn8tton21 deposirary authouity. BU D/upEsT S.'REJETV 018 E 12*i7'S12NAT1 () 1\1./EL 'RECOGMmON OF THE OEPOS ! T OF MtCROORGAM) 5MS F'THE P-U1RPC DJv Pn J ?. ftOt-li ; F : FOR THE PURPOSES OF PATENT PROCEDURE TERNATiOAL FOP.'! n nr harm. Co., LtC. ? 3-5.'Hajeo-n Pattn-myn Hwasune-Kun RECEIFF IN THE CA. 5E OF AN OMGlNAJ, ri; rons: _; i-do, issted PurSUnnt : to Ftlt7 : 7, 7 v) Blue RcpubHc or Korea INTERATiON/U, DEPOST/Y AUTHOR) 1Y identilit'. d st. the boTn of thJS pag-e L j I. fC'ENTTFiC. ATJON OF THE MJCROORGANtSM Mentfcauon refefecce ven by tbe Accessitjn number ej) by the LEPC7ST'TC ?. P< IIJTF : F2N1Tf0JllJ.. DEPQS1Tl. e. Y lUjMIrcI'rY' : t-3h110 ? . 1 TvCCt-10178 , TB2llOTO. l KCC'M-1017c° 12. SCIE !'riF : C iE. 5CRlPTIOH AMD/OR PROPOSED TAXOMOMiC DES ! GNAT) ON The micrporgBjoism ideMiRed uncte't abovt ; was accomp<anEd by : CD a scientinc descrp7ior 12 a pTOpoaed LELXOiiomk ctestgnation with Tnth a cross where appticable) TO. RECEIPT AND ACCEPTANCE "li. ; Interta, l : i. an=il Daosiy lui : lnotti ; accept. the miC1'GGrg2riism irn$inecL ttaader I : Lor'e, ulhich w. rocchtedby it on. Dec. 23. 1999 (dsM of the ongna] deposit* ru, tl'7.''EIL1IATJOJaAT DI'7SIT1F ;.'Lr vx'FJO. i'Tr Mame Hofean Culture Center oMfcroorfatusms SiE'natut'< (s) of person (s) having the power to ìcpr-esent ffie Zn ; ermationz] Deposit3ry Address : 2'1, '=n BS ltuthority of of authouizedial. s HoDgje-I-doB, 'r Seodaemun-p'nS' Seodzemun-E, u ujm Sendaemun-gu RepuMc o Korea M'c ? Ef' , _. bWi. _ 1 ere ltule s4 (u) applies, stIch ciste is the date tm whic] + the sta. tls of inct@ xsilaIy gufnorìtzT was acqtj3red-'kilere a deposit made outside the Sudapesi Treaty after the scqDiaitMn of the sh. <s cf] neernacioni clepositCy aut, horihl is convertec into a deposit der t7ze Budapest zreasYw such cI, te is 1. he ate UN whjch the nHCJ-oormsm was received by the JicnatioHHi {positsn authuutty.