KR960015746B1 - RECOMBINANT VECTOR OF HUMAN LEUKOCYTE INTERFERON Ñß-2 - Google Patents

Abstract

A plasmid vector pYAC105(VKPM B-6492) for expressing human leukocyte interferon alpha-2 and transformed Pseudomonas putida VG-86(VKPM B-6492) with pYAC105 is prepd. The human leukocyte interferon alpha-2 is prepd. by: (1) crushing Pseudomonas putida VG-86(VKPM B-6492) and centrifuging; (2) adding polyethyleneimine into the obtd. cell protein extract, centrifuging, fractioning by adding ammonium sulfate into the obtd. supernatant; (3) passing the fractioned protein through phenyl silochrome S-80 chromatography; (4) re-passing the obtd. protein through immuno affinity chromatography contg. monoclonal antibody as absorbent; and (5) eluting the human leukocyte interferon alpha-2 with 0.1M glycine buffer(pH 3.0) contg. 0.3M NaCl.

Description

Human leukocyte interferon α-2 recombinant expression vector and transformant

1 shows the nucleotide sequence of the interferon a-2 gene.

2 shows a restriction map of the pVG3 plasmid.

3 is a diagram showing the process of constructing the pAYC105 of the present invention.

4 is a photograph showing the results of SDS-PAGE analysis of the purification of interferon α-2 using immunoaffinity chromatography.

5 is a photograph showing the results of SDS-PAGE analysis of the purification of interferon α-2 using multi-step preparative chromatography.

The present invention relates to a method for producing human leukocyte interferon α-2. More specifically, the present invention relates to a method for purifying antiviral human leukocyte interferon α-2 recombinant expression vector, microorganisms transformed therewith and interferon α-2 produced therefrom.

Interferon is a protein that is biosynthesized by certain human cells in response to viral infection, and can be divided into three types depending on the production cell line and its structural and functional characteristics:

1) alpha or leucocyte interferon produced by leukocytes;

2) beta or fibroblast interferon made by fibroblasts; And,

3) Gamma or immune interferon produced by T-lymphocytes.

In addition to antiviral activity, interferon is characterized by anti-proliferative activity and affects the immune response, which makes it possible for drugs prepared based on interferon to be effectively used to treat malignant tumors.

To date, highly purified human interferon preparations have been provided, but leukocyte interferon isolated from the blood of recipients has not been provided in sufficient amounts for clinical wide application. The reason for this is that a single effective dose of interferon should be at least 10 ⁶ AU in clinical trials, but one liter of blood usually contains less than 10 ⁵ AU. to be.

In the present invention, to produce a large amount of human leukocyte interferon α-2, the recombinant expression vector and the production strain transformed therefrom were cultured with aeration in the presence of antibiotics in a neutral medium containing carbon, nitrogen, inorganic salts and growth promoters. Next, the final product was separated.

In the present invention, Pseudomonas sp. VG-86 transformed with plasmid pAYC105 was used as a production strain, and the strain is a genetic and industrial microbiological research institute which is an international depository institution located in Russia on August 2, 1993. Deposited under the accession number VKPM B-6492 to Genetics and Industrial Microorganism Breeding (VNII Genetika). The above-mentioned strains are cultured in the presence of antibiotics such as streptomycin or tetracycline, preferably in the presence of 30-50 mg / 1 tetracycline and 50-150 mg / 1 streptomycin.

By using Pseudomonas strain VG-86, a novel production strain of the present invention, it was possible to increase the yield of the final product of human leukocyte interferon α-2 and to be effectively isolated and purified through a simple separation process. The novel Pseudomonas strain VG-86 of the present invention is cloned as part of the multicopy plasmid pAYC105 under the control of the D gene, which is the regulatory region of bacteriophage φX174.

Pseudomonas sp. VG-86 of the present invention is a hybrid of a plasmid vector pAYC34 and pIFN-α2-P2 plasmid, which combines a replicon of pBR322 and an interferon α-2 gene, wherein the transformed cells are tetracycline It is made to be resistant to ampicillin. The interferon α-2 gene present in plasmid pIFN-α2-P2 is controlled by bacteriophage øX174 modulator D. On the other hand, plasmid vector pAYC34 consisting of 9400 base pairs is linked to Inc p4 / Q, an incompatibility group, and makes transformed cells resistant to streptomycin.

On the other hand, plasmids pAYC34 and pIFN-α2-P2 DNA are processed and linked with restriction enzyme PstI and transformed into E. coli strain C600 (E. coli C600). Transformants are selected, for example, in a complete nutrient medium containing antibiotics of 50 μ / ml ampicillin, 100 μ / ml streptomycin and 25 μ / ml tetracycline. Plasmid DNA, designated pVG3, was selected from the transformant clones, which was confirmed by hybridization of plasmids pAYC34 and pIFN-α2-P2 based on size and restriction map. Plasmid pVG3 confers transformed cells cell resistance to streptomycin like pAYC34 and tetracycline and ampicillin like pIFN-α2-P2. In addition, since the plasmid pVG3 has 14800 base pairs, it can be seen that the plasmid pVG3 consists of 5400 base pairs plasmid pIFN-α2-P2 and 9400 nucleotide pairs pAYC34.

Escherichia coli transformed with plasmid pVG3 was grown in fully neutral medium without antibiotics, resulting in the deletion of markers showing that some bacteria are resistant to ampicillin. One plasmid DNA isolated from non-viable clones in the presence of ampicillin was named pAYC105 (B-6492), which has a molecular weight of 12.7 kb and includes each of the following fragments:

1) Pst1-EcoR1 fragment (7.7 kb) from RSF1010 having a gene resistant to streptomycin;

2) EcoR1-BamH1 fragment (0.38 kb) from pBR322;

3) BamH1-HindIII fragment (3.16 kb) from pBR322 having a gene resistant to tetracycline; And,

4) HindIII-Pst1 fragment (1.45 kb) carrying the alpha interferon gene under the control of the D gene of bacteriophage øX174.

Plasmid pAYC105 comprising such fragments determines resistance to streptomycin and tetracycline in transformed cells.

Conjugative wide-host range plasmid R751 was inserted into one of E. coli C600, including the hybrid plasmid pAYC105, in which plasmid pAYC105 is a bacterium in the genus S. typhimurium and other Gram-negative bacteria. This is possible through a series of conjugative crosses that are translated into. Then, transconjugant bacteria were obtained in the selection medium containing each of the antibiotics described above, and the ability to synthesize interferon was tested.

The cells obtained by the above method were cultured under aeration, and the temperature conditions were 30 ° C. for 6 to 10 hours until the cell density became 2 to 5 × 10 ⁹ cells / ml in L-broth medium. It was incubated with stirring at. The bacteria were harvested by centrifugation and pulverized by ultrasonication, and the amount of interferon present in the cell extract was measured by radioimmunoassay. Eighteen strains of Gram-negative bacteria associated with the genus Esccherichia, Salmonella, Pseudomonas, Methylomonas, Erwinia, and Rhizobium were tested. Most of the strains have been found to produce active interferon. The highest productivity was Pseudomonas sp. VG-86 strain (Pseudomonas sp. VG-86), under the above-described conditions, it was confirmed that the strain produces 5 × 10 ⁹ to 1 × 10 ¹⁰ AU of interferon per liter of culture.

On the other hand, the growth conditions and morphological characteristics of Pseudomonas VG-86 strain (VKPM B-6492) carrying the plasmid pAYC105 described above are as follows:

TABLE 1

Hereinafter, the present invention will be described in more detail.

Pseudomonas VG-86 strains were incubated for 8 to 12 hours in an agarod-ped nutrient medium at a temperature of 28 to 30 ° C. and inoculated again in liquid nutrient medium, and the inoculum was inoculated for 3 to 6 hours at 28 ° C. Incubate under vigorous stirring and aeration. Liquid nutrient media containing carbon, nitrogen, inorganic salts and growth factors, for example glucose, yeast extract, autolysate, tryptone, peptone, hydrolysates of casein or yeast, or mixtures thereof It is appropriate to incubate in the incubator of the medium in the presence of streptomycin or tetracycline or both antibiotics, preferably in the presence of 30 to 50 mg / 1 of tetracycline and 50 to 150 mg / l of streptomycin. Fermentation is carried out under vigorous aeration for 6 to 10 hours at 28-32 ° C. medium of pH 6.7-7.1. During the fermentation, the absorbance at ₅₅₀ hm of the bacterial suspension (A ₅₅₀ ) is measured. At the end of the fermentation the OD value reaches approximately 5 or 7 optical units. The yield of interferon was determined by measuring radioimmunoassay or antiviral activity against human fibroblast culture under the aforementioned culture conditions, containing 5 × 10 ⁹ to 1.5 × 10 ¹⁰ AU per liter of culture. .

When the fermentation is complete and the OD value described above is reached, the bacterial cells are collected and resuspended in an appropriate buffer solution by centrifugation, and subjected to sonication or other method, lysozyme treatment, glass or metal spheres. using a ball and a French press to decompose the cells or destroy the cell membrane.

Then, cell debris is separated by centrifugation, and a cell lysate buffer containing 15-20% saccharose and a protein inhibitor at pH 7.5 to 8.2 and 3 to 5 ° C is used. Cells are destroyed by the used lysis. The nucleic acid and the insoluble constituents of the cell are removed using a polyethylene imine at a final concentration of 0.25 to 0.35%, and the protein is fractionated using ammonium sulfate having a saturation of 70 to 85% at pH 7.2 to 7.9. . In addition, the protein is fractionated by using hydrophobic chromatography of phenyl silochrome S-80, which is an adsorbent having a phenyl group.

In this way, the fractionated electric protein can be purified by the following two purification methods.

I. Immuno affinity chromatography method

Insoluble proteins are dispersed in buffer and human interferon a-2 is isolated using monoclonal antibody type 5A6 immunoaffinity chromatography. Purification of human interferon α-2 by immunoaffinity chromatography destroys cells in hemolytic buffer, which allows the scale of the process and significantly shorten lysis time.

II. Multistep Preparative Chromatography Method

35% phosphoric acid solution is treated until pH 4.5 to 5.0 to fractionate the protein according to the hydrogen ion concentration, and the protein solution is concentrated and diafiltration. In addition, the protein was fractionated by ion exchange resin chromatography using cellulose DE-52 (Whatman) and cellulose SM-52, and then concentrated to gel the protein solution by Sephadex G-100 (Pharmacia, Sweden). Filtration (gel filtration).

The main difference between the purification method using the multistage purification chromatography and the purification method using the immunoaffinity chromatography described above is that the ion exchange resin using cellulose DE-52 and cellulose SM-52 instead of immunoaffinity chromatography. Chromatography and gel filtration using Sephadex G-100 are all performed at least 30 times.

Thus, the yield of purified interferon was 20 to 60%, the purification rate was 200 to 500 times, the titer of the purified product and the product was confirmed to be about 4 × 10 ⁸ AU, the purification process is vesicular stomatitis By measuring the antiviral activity of human fibroblast cultures with a virus, it can be traced by radioimmunoassay or Enzyme-Linked Immunosorbent Assay (ELISA). Purified interferon can be assayed not only by examining the composition of amino acids and the N-terminal amino acids of the final product separated, but also by immunological methods, gel filtration and precipitation techniques by centrifugation. The homogeneous human leukocyte interferon α-2 obtained in this way was confirmed to have structural and functional characteristics specific to the interferon of this type.

Hereinafter, the present invention will be described in more detail with reference to Examples.

These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples according to the gist of the present invention.

Example 1

Preparation of pVG3 Plasmid Vector

MRNAs of interferon α-2 were isolated from human leukocytes infected with Newcastle disease according to Manniatis et al. [Maniatis T., Fritsch EE, Sambrook J. Molecular Cloning: A laboratory Manual, Cold Spring Harbor Laboratory, 1982 .] The obtained Ploy [A] ⁺ -RNA was converted to cDNA using reverse transcriptase, and the second strand was synthesized with DNA polymerase I (Klenow fragment). The DNA thus obtained was linked to dCTP in the presence of terminal transferase and to the PstI position of plasmid pBR322 to which poly [dG] was attached. This gene was then transformed into E. coli and selected for Ap ^S -Tc ^R clones that were sensitive to ampicillin and resistant to tetracycline. Two clones encoding the N-terminus and C-terminus of interferon α-2 were identified, respectively, and named pIFN-α2-504 and pIFN-α2-0, respectively.

Obtained by connecting pIFN-α2-504 DNA obtained by cleaving the entire gene of interferon α-2 with Sau3A and PvuII to a Sau3A-EcoRI adapter and linking the hydrolyzed pIFN-α2-0 DNA with EcoRI-PvuII It was. A fragment encoding the interferon α-2 gene was linked to the EcoRI position of pBR322, and the resulting plasmid was named pIFN-α2-310, and the plasmid was partially cleaved with EcoRI to be hydrolyzed to nuclease S1. Decompose and reconnect. Clones with the desired fulllasmid were selected and named pIFN-α2-310.

The Alu I fragment of øX174 phage encoding the D promoter was isolated and linked to pIFN-α2-310 DNA treated with the hydrolase S1 and EcoRI. E. coli was transformed to isolate a plasmid with the desired structure and named it pIFN-α2-P2.

To produce interferon α-2 in Pseudomonas putida's medium, plasmid pIFN-α2-P2 was cleaved with PstI and linked to plasmid pAYC37 with a broad host range cut with PstI. The resulting plasmid was named pVG3, which can be replicated in Pseudomonas putida, which is resistant to Ap, Tc and Sm. FIG. 1 shows the nucleotide sequence of the interferon α-2 gene, FIG. 2 shows the restriction map of the pVG3 plasmid, FIG. 3 shows the construction of pVG3 plasmid from pAYC34 and pIFN-α2-P2 and the construction of pAYC105 therefrom. Is shown schematically.

Example 2

Preparation of pYAC105 Plasmid Vector and Transformant

Escherichia coli transformed with pVG3 prepared in Example 1 was grown in a complete neutral medium without antibiotics to select a 12.7 kb plasmid lacking an ampicillin-resistant marker, which was named pAYC105. 3 degrees]. pAYC105 is a Pst1-EcoR1 fragment (7.7 kb) from RSF101 having a gene resistant to streptomycin, each of the following gene fragments: EcoR1-BamH1 fragment (0.38 kb) from pBR322; BamH1-HindIII fragment (3.16 kb) from pBR322 having a gene resistant to tetracycline: and each fragment of the HindIII-Pst1 fragment (1.45 kb) carrying the alpha interferon gene under the control of the D gene of bacteriophage? X174. Plasmid pAYC105 comprising such fragments confers resistance to streptomycin and tetracycline on transformed cells.

Plasmid pAYC105 was transformed into Pseudomonas putida VG-86 according to a known transformation method, and the strain was carried out on 2 August 1993 by the Institute for Genetic and Industrial Microorganisms, an international depository institution. Deposited under the accession number VKPM B-6492 to Genetics and Industrial Microorganism Breeding (VNII Genetika). The above-mentioned strains are cultured in the presence of antibiotics such as streptomycin or tetracycline, preferably in the presence of 30-50 mg / 1 tetracycline and 50-150 mg / 1 streptomycin.

Example 3

Culture of Pseudomonas VG-84 (Ⅰ)

Pseudomonas putida VG-86 strain prepared in Example 2 was incubated for 12 hours at a temperature of 28 ℃ in a solid agar medium having the composition shown in Table 2 below:

TABLE 2

Cells incubated in an electric agar medium were transferred to a 750 ml Erlenmeyer flask containing 100 ml of culture medium without agar, incubated for 6 hours with vigorous stirring at 240 rpm at a temperature of 28 ° C, and then A nutrient medium (pH 6.7 to 6.9) with the above composition was added at a rate of 5% and grown for 6 hours under vigorous stirring and aeration at a temperature of 28 ± 0.5 ° C. Since the aeration and agitation conditions were selected, the dissolved oxygen concentration of the grown culture medium could not be limited. For this purpose, the partial pressure of oxygen was maintained at a level of 5 to 10% of the saturated oxygen concentration. In order to maintain a constant pH value, ammonia water was used as a buffer solution, and cell growth was tracked by measuring the OD value of the culture solution. The absorbance was measured every hour to stop fermentation when OD A ₅₅₀ reached 5 optical units.

Further fermentation resulted in a titer of human leukocyte interferon α-2 of 1.5 × 10 ¹⁰ AU per liter of culture. In order to measure the concentration of the interferon thus obtained, a solid phase modification of RIA using an antiinterferon rabbit polyclonal antibody and a mouse monoclonal antibody labeled with a ¹²⁵ I isotope was used. In addition, antiviral interferon activity was measured by the protective effect against the cytopathic action of vesicular stomatitis virus prepared in the culture of human diploid fibroblasts. As a reference, standard interferon MRCB69 / 19 (Great Britain) was used.

Example 4

Culture of Pseudomonas VG-84 (Ⅱ)

Production strains were cultured in the same manner as in Example 3, except that the production strains were grown in the presence of an antibiotic of 50 mg / 1 of streptomycin and 30 mg / tetracycline. After six hours of fermentation, the bacterial suspension was 4.2 optical units and one liter of culture contained 7.5 × 10 ⁹ AU of human leukocyte interferon α-2.

Example 5

Culture of Pseudomonas VG-84 (Ⅲ)

Production strains were cultured in the same manner as in Example 2 except that the growing strain was grown in the presence of 50 mg / 1 of the antibiotic tetracycline. The bacterial suspension OD after 5.5 hours of fermentation was 5.5 optical units and the interferon concentration per liter of culture was 9.5 × 10 ⁹ AU.

Example 6

Purification by immunoaffinity chromatography

6.1. Cell destruction

715 g of Pseudomonas putida VG-86 of human interferon α-2 frozen at −70 ° C. were ground to pieces of approximately 1 to 2 cm 3, and then hemolytic buffer solution (0.1 M-Tris.HC1, 15% saccharose, 5 mM) EDTA, 0.5M sodium chloride, 10 mM phenylmethylsulfonylfluoride, 0.05% thiodiglycol, pH 7.9) 3575 ml were added, and placed in a Simax reactor and mixed for 1 hour at 4 ° C. The destroyed cells were centrifuged at 10,000 rpm for 1 hour using a centrifuge (Beckman, J2-21, U.S.A.), and 3,700 ml of intracellular protein extracts were obtained by this method.

6.2. Treatment with Polyethylenimine

To obtain intracellular protein extract, 190 cm 3 of 5% polyethyleneimine solution was added to a final concentration of 0.25%, followed by centrifugation for 1 hour. The suspension was centrifuged for 30 minutes at 10,000 rpm with a JA-10 rotor in a centrifuge to obtain 3,800 ml of supernatant.

6.3. Fraction by Ammonium Sulfate

Ammonium sulfate was added and dissolved to 85% saturated concentration while maintaining pH 7.2 to 7.9 in the supernatant obtained in a Simax reactor (capacity 101liters). The mixture was mixed for 2 hours or overnight to allow formation of a large amount of protein mass and centrifugation was used to dissolve all the precipitates with 4,500 cm 3 of 0.02 M phosphate buffer containing 3 M sodium chloride. This gave 5,000 cm 3 human interferon a-2 solution.

6.4. Hydrophobic Chromatography on Phenyl Silochrome S-80

The protein solution obtained in 6.3. Was charged with 300 ml of phenyl silochrome S-80 and poured onto a column K100 / 45 (Pharmacia, Sweden) equilibrated with 0.02 M phosphate buffer (pH 6.5) containing 3 M sodium chloride. . The eguilibrating solution was then passed through the column until it reached the basic line of the control device for protein detection (UV-1, Pharmacia, Sweden). Fractions of human interferon α-2 were eluted under sterilized with System Pelicon (Milipore Compant, U.S.A.) 0.02M phosphate buffer (pH 6.5). Elution rate on the column was maintained at 4 L / h, thereby obtaining a fraction of human interferon α-2. In this process, the adsorbent was regenerated by ten volumes of water, 35% isopropyl alcohol, 75% isopropyl alcohol, 35% isopropyl alcohol and water.

6.5. Immunoaffinity Chromatography on Adsorbents with Monoclonal Antibodies 6.5.1. Preparation of Immunosorbents

The suspension obtained by dissolving 171 g of activated Sepharose in 1 L of 0.001 M HC1 and mixing for 2 to 3 minutes was poured onto a glass filter, and then washed with 39.3 L of 0.001 M sodium chloride solution. 2.88 g of lyophilized monoclonal antibody 5A6 was dissolved in 576 cm 3 of 0.1 M sodium acetate solution (pH 8.3), and 0.5 M sodium chloride was added and mixed using a mechanical mixer for about 2 hours at 100 rpm at ambient temperature. . After mixing, the suspension was poured onto a glass filter and the solution was taken out. Then, the adsorbent was treated with 1 L of 1M ethanolamine solution (pH 8.0) and mixed for 2 hours under the same conditions at circulation temperature. The adsorbent was poured onto a glass filter and the solution was withdrawn and 0.5 M sodium chloride dissolved in 1 L of 0.1 M sodium acetate solution. The suspension of added immunosorbent is poured into a glass filter, washed with 5 L of 0.1 M sodium acetate solution (solution pH 8.37) with 0.5 M sodium chloride, and then A solution (8.0 g / L sodium chloride, 0.2 g / L potassium chloride). , 1.15 g / L sodium hydrophosphate, 0.2 g / L potassium dihydrophosphate, pH7.2), and then washed with 2 L of A solution added with 0.002% thyresol. At this time, the degree of binding of the monoclonal antibody to the adsorbent was 0.95.

6.5.2. Purification by immunoaffinity chromatography

The adsorbent (500 ml) obtained by the above 6.5.1. Was poured onto column K100 / 45 (Pharmacia, Sweden) and washed with 3 L of solution A at a rate of 2 L / hour. 500 ml of the protein solution obtained in the above step 6.4 was poured onto the column at the same speed, and the column was washed in the following order: A solution 1.5L → 0.5M sodium chloride 5L → A solution 1.5L. Isolation of human interferon α-2 attached to the immunosorbent was performed using 0.1 M glycine solution (pH 3.0) containing 0.3 M sodium chloride. The separated proteins were collected into separate fractions by a detector of a fraction collector FRAC-300 (Pharmacia, Sweden), and 0.91 L was obtained by collecting the fractions containing interferon α-2 in one place. Regeneration of immunosorbents with monoclonal antibodies was accomplished by washing with 3 L of 0.15 M sodium chloride solution followed by 5 L of A solution.

4 is a photograph showing the results of SDS-PAGE analysis of the purification of interferon α-2 using immunoaffinity chromatography, wherein lane 1 is a molecular weight marker; The second lane comprises cell protein crude extract; The third lane comprises a protein fraction isolated from a phenyl silochrome S-80 column; Lane 4 represents pure purified interferon obtained by immunoaffinity chromatography.

Example 7

Purification by Multistep Preparative Chromatography

7.1. Cell disruption

4268 g of cell mass of Pseudomonas putida VG-84 frozen at −70 ° C. were crushed into pieces of 1 to 2 cm 3 and placed in a Simax reactor: Hemolytic buffer (0.1 M Tris-HC1, 15). 21,340 ml of% Saccharose, 5 mM EDTA, 0.2 M sodium chloride, 10 mM phenylmethylsulfonylfluoride, 0.005% thiodiglycol, pH 7.9) was added and mixed at 4 ° C. for 1 hour. The cells so lysed were centrifuged at 10,000 rpm / min to obtain 22,100 ml of intracellular protein extract.

7.2. Polyethyleneimine Treatment

1,140 ml of 5% polyethyleneimine solution was added to the protein extraction solution so that the final concentration of polyethyleneimine in the total solution was 0.25. The suspension was then centrifuged for 30 minutes with a rotor JA-10 in a centrifuge (Beckman J2-21, U.S.A.) at 10,000 rpm / min to give 23,200 ml of solution as supernatant.

7.3. Fraction by Ammonium Sulfate

The supernatant obtained in Example 7.2 above was placed in a SYMAX reactor (capacity 50L), and then ammonium sulfate was added to 85% saturated concentration and mixed for 2 hours. At this time, the pH was adjusted to 7.2 to 7.9, and the precipitate was collected by centrifugation again, and then dissolved in 16,000 ml of 0.025 M phosphate buffer (pH 7.1) to which 3 M sodium chloride solution was added. Thus, 19,400 ml of human interferon α-2 solution was obtained.

7.4. Hydrophobic Chromatography on Phenyl Silochrome S-80

The protein solution obtained above was fractionated by chromatography of 3,000 ml of phenyl silochrome S-80 using column K100 / 45 (Pharmacia, Sweden) equilibrated with 0.025 M phosphate buffer (pH 7.1) with 3 M sodium chloride. . All procedures at this stage were performed sterile.

7.5. Fraction using pH

35% phosphoric acid solution was added to 12,400 ml of protein solution in a SYMAX reactor (volume 20L) until pH 4.75, followed by mixing for 1 hour. The suspension thus obtained was centrifuged for 30 minutes using a rotor JA-10 of a centrifuge (Beckman J 2-21, U.S.A.) at 10,000 rpm / min to finally collect 11,920 ml of supernatant.

7.6. Concentration and Diafiltration of Protein Solutions

Concentration and dialysis of the protein solution was carried out using a cassette of filters (PTGC type) having a transmission limit of 10,000 Daltons on a pelicon type system (Millipore, USA). The protein solution was concentrated to 1,200 ml (pH 8.0) and then 2,000 ml of 0.01 M phosphate buffer was added. Then, six dialysiss were performed, and the pH of 1,2000 ml of the final concentrate was 8.00.

7.7. Ion Exchange Resin Chromatography with Cellulose DE-52

Protein solution was fractionated on a Bioprocess 113 (Pharmacia, Sweden) column containing 4,300 ml of cellulose DE-52 (Whatman, Great Britain) balanced with 0.01 M phosphate buffer (pH 8.0). Human α-2 interferon fractions were eluted with 0.05M acetic acid buffer (pH 5.0).

7.8. Ion Exchange Resin Chromatography with Cellulose SM-52

Protein solution 3,200 ml obtained above using column K100 / 45 (Pharmacia, Sweden) containing 3,000 ml of cellulose SM-52 (Whatman, Great Britain) equilibrated with 0.1 M acetic acid buffer (pH 5.0) Was chromatographed. The human α-2 interferon fraction was eluted by flowing 0.1 M acetic acid buffer and 0.1 M acetic acid buffer containing 0.4 M sodium chloride (pH 5.0) in a linear gradient at 2 L / h.

7.9. Concentration of Protein Solution

Concentration of the protein solution was carried out using a cassette of filters (PTGC type) having a permeation limit of 10,000 Daltons on a Minitan type system (Millipore, U.S.A.), and the final volume obtained was concentrated to 400 ml.

7.10. Gel Filtration with Sephadex G-100

In a column K100 / 100 containing 7,500 ml of Sephadex G-100 (Pharmacia, Sweden) equilibrated with 0.1 M phosphate buffer (pH 7.2) with 0.2 M sodium chloride, 300 ml / Gel filtered at a rate of h. Fractions containing homogeneous human α-2 interferon were collected in one place. The yield of homogeneous human α-2 interferon was 13.1% and the activity was 1.45 × 10 ¹⁰ IU / protein 1 mg.

5 is a photograph showing the results of SDS-PAGE analysis of the purification of interferon α-2 using multi-step preparative chromatography, wherein lane 1 is a molecular weight marker; The second lane is an intracellular protein extract; The third lane comprises a protein fraction isolated from a phenyl silochrome S-80 column; Lane 4 is a protein obtained after the PH fractionation at PH 4.5; Lane 5 is a protein obtained after concentration with a filter at PH 8; Lane 6 is an interferon fraction isolated from cellulose DE-52 column; Lane 7 is an interferon fraction isolated from cellulose CM-52 column; Lane 8 was pure interferon eluted with Sephadex G-100 column; Lane 9 represents sterile filtered interferon as the final product.

The electrophoretic homogeneous human leukocyte interferon α-2 obtained in Examples 6 and 7 described above showed a purification rate of 210, a yield of 41% in terms of activity, and a specific activity of 4.0 × 10 ⁸ AU. In particular, the human interferon α-2 isolated in the present invention was no different from human leukocyte α-2 isolated from other bacterial producing strains with respect to specific activity, molecular weight (18.4 KD), amino acid composition and other properties. In particular, as a result of Edman automatic N-terminal sequencing, homogeneous interferons isolated from Pseudomonas VG-86 production strains were identified as Cys-Asp-Leu-Pro-Glu-Thr-His-Ser-. Characterized by the N-terminal amino acid sequence of, the N-terminal did not contain a methionine residue. Thus, it was confirmed that the above interferon is exactly the same as the interferon α-2 produced by human leukocytes.

As described and demonstrated in detail above, the present invention provides an expression vector capable of obtaining the same amount of interferon with less biomass with high activity by using a novel highly productive strain, a microorganism transformed therefrom and the same. Provided are methods for preparing human α-2 interferon. According to the method for producing human α-2 interferon of the present invention, it will contribute to reducing the cost of separating all the continuous processes and biomass involved in preparing a homogeneous product in the preparation of interferon.

Claims (10)

Plasmid vector pYAC105 (VKPM B-6492) expressing human alpha interferon a-2. Pseudomonas putida VG-86 (VKPM B-6492) transformed with pYAC105 of claim 1. (Iii) destroying and centrifuging Pseudomonas putida VG-86 (VKPM B-6492); (Ii) centrifugation by adding polyethyleneimine to the cell centrifuged cell protein extract, followed by fractionation by adding ammonium sulfate to the obtained supernatant; (Iii) phenyl silochrome S-80 chromatography of the electrically fractionated protein; And (iii) immunoaffinity chromatography of the electrically fractionated protein on an adsorbent having a monoclonal antibody. The method of claim 3, wherein the immunoaffinity chromatography is eluted with 0.1 M glycine buffer (pH 3.0) containing 0.3 M sodium chloride. (Iii) destroying and centrifuging Pseudomonas putida VG-86 (VKPM B-6492); (Ii) centrifugation by adding polyethyleneimine to the cell centrifuged cell protein extract, followed by fractionation by adding ammonium sulfate to the obtained supernatant; (Iii) chromatographically fractionating the previously fractionated protein with S-80, pH fractionating, concentrating and dialysis; (Iii) cellulose DE-52 ion exchange chromatography of the electrically fractionated protein; (Iii) cellulose SM-52 ion exchange chromatography of the electrically fractionated protein; And (iii) Sephadex G-100 gel filtration chromatography of the electro-fractionated protein. 6. The method of claim 5, wherein the concentration and dialysis use a PTGC type cassette having a transmission limit of 10,000 Daltons. The method according to claim 5, wherein the cellulose DE-52 ion exchange chromatography is eluted using 0.04 to 0.06 M acetic acid buffer (pH 5.0). 6. The cellulose SM-52 ion exchange chromatography of claim 5 is a linear gradient using 0.1M acetic acid buffer solution (pH 4.8 to 5.2) containing 0.1M acetic acid buffer solution and 0.3 to 0.5M sodium chloride. Eluting with water. Human alpha interferon α-2 prepared from Pseudomonas putida VG-86 (VKPM B-6492) according to the method of claim 3. Human alpha interferon α-2 prepared from Pseudomonas putida VG-86 (VKPM B-6492) according to the method of claim 5.