DE69232094T2 - Recombinant gene coding for alpha interferon and expression vector therefor - Google Patents

Description

Field of the invention

The present invention relates to a recombinant gene encoding human alpha interferon and its expression in yeast. More particularly, the invention relates to a recombinant gene encoding human alpha interferon suitable for mass production of human alpha interferon in yeast, an expression vector therefor, yeast cells transformed with the expression vector, a process for producing human alpha interferon by using the transformed yeast cells, and a process for purifying the alpha interferon produced in yeast cells in high yield.

Presentation of the state of the art

Interferon, a cytokine, is a protein with antiviral activities produced by animal cells in response to viral infection. Interferons are classified based on their origin: alpha interferon (IFN-α), also known as leukocyte interferon, is produced mainly in B lymphocytes, null lymphocytes or macrophages; beta interferon (IFN-β), also known as fibroblastic interferon, is produced mainly by fibroblasts or epidermal cells; and gamma-interferon (IFN-γ), also known as immune interferon, is mainly produced by T lymphocytes with macrophages (Stanton, G. J. et al., Tex. Rep. Biol. Med. 41, 84-88 (1981); Kirchner, H. et al., Tex. Rep. Biol. Med. 41, 89-93 (1981)).

To date, 20 or more types of alpha-interferon genes have been identified; most of them encode 165 or 166 amino acids.

The first alpha interferon used in a clinical trial was one produced in buffy coat leukocytes stimulated by Sendai virus; its purity was not more than about 1% (Cantell, K. and Hirvonen, Tex. Rep. Biol. Med. 35, 138-144 (1977)).

Recently, human alpha interferons produced by recombinant DNA technology have been clinically tested. As a result, they have been reported to be effective in the treatment of a number of cancers, particularly bladder cancer (Torti, F. M. et al., J. Clin. Onco. 3, 506-512 (1985)) and kidney cancer (Vugrin, D. et al., Cancer Treat. Rep. 69, 817-820 (1985)), and further in the treatment of hepatitis C (Davis, G. G. et al., N. Engl. J. Med. 321, 1501-1506 (1989); Bisceglie, A. D. et al., N. Engl. J. Med. 321, 1506-1510 (1989)).

The production of human alpha interferons using recombinant DNA technology is generally limited to those methods that use E. coli as host cells (Nagata, S. et al., Nature 284, 316-320 (1980); Goeddel, D, V. et al., Nature 287, 411-416 (1980); Streuli, M. et al., Science 209, 1343-1347 (1980); Goeddel, D. V, et al., NAR 8, 4057-4074 (1980); Dreynck, R. et al., Nature 287, 193-197 (1980)). Alpha interferons produced in this way may not be identical to the natural product because E. coli cannot glycosylate proteins produced in it; and native E. coli proteins, such as endotoxin, may remain in the alpha interferons even after they are purified.

In this context, yeasts have several advantages over E. coli as a host cell for the production of alpha-interferons, namely: the mechanism of glycosylation in yeast is similar to that in animal cells; and furthermore, yeasts do not produce substances harmful to humans, as is evident from the fact that yeasts have been used in the food processing industry for several centuries.

However, when interferons are produced in yeast cells using recombinant DNA technology, it is always critical to purify the interferons from the native yeast substances for clinical use.

Alpha interferons produced in E. coli or yeast cells are purified to high purity using reversed-phase high-performance liquid chromatography (Rubinstein, M. et al., Arch. Biochem. Biophys. 210, 307-318 (1981)) or monoclonal antibody affinity chromatography (Berg, K. and Heron, I., Methods Enzymol. 78, 487-499 (1981)).

Needless to say, the bioactivity of interferons is important to be clinically useful. In this context, natural alpha-interferon has two disulfide bonds, i.e. one between two cysteines, which are the 1st and 98th amino acids, and another between another pair of cysteines, which are the 29th and 138th amino acids. However, most of the alpha-interferon produced using E. coli or yeast is in a reduced form, which does not have the said disulfide bonds, or as an incompletely oxidized form with only one of the said disulfide bonds, both of which are denatured forms. Furthermore, they often form oligomers, such as dimers, by bonding between them during a purification process.

Therefore, numerous efforts have been made to obtain alpha-interferon in an active form and in a high degree of purity.

For example, Korean Patent Application 90-13450 discloses a method comprising the following steps: dissolving alpha-interferon in a denatured form purified from a host cell using a conventional method in a solution containing a reducing agent such as β-mercaptoethanol and dithiothreitol; refolding the reduced alpha-interferon and then isolating the refolded alpha-interferon by anion exchange chromatography to obtain alpha-interferon with high purity and activity. However, this method has the defect that refolded but inactive alpha-interferon proteins, e.g., incompletely oxidized proteins or dimers, remain in the final product.

If cation exchange chromatography is additionally used, the inactive alpha-interferon proteins can be removed. However, such removal is not helpful in increasing the yield of active alpha-interferon.

EP-A-0 128 467 describes a DNA sequence for rINF-α, in which at least one triplet encoding a cysteine is replaced by a triplet encoding an amino acid incapable of forming an intermolecular disulfide bond. EP-A-0 100 561 discloses the expression of unmodified human interferon in yeast.

Summary of the invention

It is an object of the present invention to provide a recombinant gene (rhIFN-α gene) encoding human alpha interferon for efficient expression in yeast.

It is a further object of the present invention to provide an expression vector comprising the rhIFN-α gene and yeast cells transformed with the expression vector.

It is a further object of the present invention to provide a process for producing human alpha interferon (hIFN-α) using transformed yeast.

Accordingly, according to one aspect of the present invention, there is provided a rhIFN-α gene having a nucleotide sequence shown in Figure 1, which is designed to produce mature hIFN-α in yeast.

According to a further aspect of the present invention, there is provided an expression vector capable of functioning in yeast and comprising the rhIFN-α gene, as well as yeast cells transformed with the vector.

According to another aspect of the present invention there is provided a method for producing hIFN-α in yeast, which comprises culturing the transformed yeast under culture conditions suitable for expression of the rhIFN-α gene.

Short description of the drawing

Figures 1A and 1B show the nucleotide sequence of a rhIFN-α gene of the present invention and the amino acid sequence encoded thereby, as well as the nucleotide sequence of natural hIFN-α cDNA prepared using an isolated human mRNA and the amino acid sequence encoded thereby, respectively;

Fig. 2 shows the nucleotide sequence of the AG885 promoter;

Figures 3A and 3B describe the strategies for the construction of two expression vectors containing the rhIFN-α gene;

Figures 4A and 4B reveal the results of electrophoresis of cell extracts of the transformed yeast cultured under conditions suitable for the expression of the rhIFN-α gene;

Fig. 5 shows the result of CM-Sepharose column chromatography of the protein sample from the transformed yeast;

Figure 6 presents the results of non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the dimers obtained using CM-Sepharose column chromatography and the monomers converted from the dimers using a redox agent; and

Figure 7 provides the result of C18 reversed-phase high performance liquid chromatography for the monomers obtained using CM-Sepharose column chromatography and the monomers converted from the dimers using a redox agent.

Detailed description of the preferred embodiments

All literature references cited here are hereby incorporated by reference in their entirety into the subject matter of the present application.

As used herein, the following terms shall have the following meanings:

The term "active form of human alpha interferon" or "active hIFN-α" refers to a monomeric human alpha interferon with two disulfide bonds, i.e. one between the two cysteines which are the 1st and 98th amino acids and another between the two cysteines which are the 29th and 138th amino acids.

The term "inactive form of human alpha interferon" or "inactive hIFN-α" refers to all human alpha interferons that do not have two disulfide bonds as described above, including those in oligomeric form, incompletely oxidized form with one disulfide bond, and with misbonded disulfide bonds.

All sequenced yeast genes, especially highly expressed genes, show a strong preference for 25 codons among the 61 possible codons; and the degree of preference for these 25 preferred codons in each gene correlates with the concentration of its mRNA in the cell (Bennezent J. L. and Hall B. D., The Journal of Bio. Chem. 257, 3026-3031 (1982)). Genes that are highly expressed show a higher preference than genes with a lower level of expression.

Furthermore, it is well known that the efficiency of transcription and translation of a gene, which is an important factor for the expression of a gene, decreases when a secondary structure of the mRNA exists.

Based on the above facts, the rhIFN-α gene of the present invention was designed to have codons preferred by yeast and to minimize the formation of a secondary mRNA structure by replacing the region corresponding to 80 base pairs in the 5'-end region of hIFN-α cDNA prepared using mRNA isolated from human cells with a new one, so that the transcription and translation efficiency of the rhIFN-α gene in a yeast cell was increased.

Furthermore, for convenience in genetic engineering, the recognition sites of four different restriction endonucleases were introduced into the rhIFN-α gene: a XbaI site (5'- TCTAGA-3') is located at the codons for the 12th and 13th amino acids, Ser and Arg; a HindIII site (5'-AAGCTT-3') was located at the codons for the 25th to 27th amino acids, Ile, Ser and Leu; BaIII (5'-AGATCT-3') was located at the codons for the 63rd to 65th amino acids, Glu, Ile and Phe; and SacI (5'-GAGCTC-3') was located at the codons for the 88th and 89th amino acids, Glu and Leu.

An initiation codon ATG is introduced immediately before the codon for the N-terminal amino acid of mature α-interferon, Cys; and two termination codons, 5'-TAA-3' and 5'-TAG-3', were provided immediately after the codon for the C-terminal (166th) amino acid, Glu. For convenience in cloning, a suitable recognition site(s) for a restriction endonuclease may be introduced at the position adjacent to the termination codons and/or before the initiation codon ATG.

The rhIFN-α gene of the present invention, which was prepared to satisfy the above requirements, has a nucleotide sequence as described in Fig. 1A. Fig. 1A also shows the amino acid sequence of the polypeptide encoded by this gene.

The expression vector according to the invention for expressing the rhIFN-α gene can be produced by using various expression systems that perform their function in yeast.

According to a preferred embodiment, there is provided an expression vector prepared by cloning the rhIFN-α gene into a vector comprising a GAP promoter. More specifically, the expression vector is prepared using a plasmid pYLBC-GAP-HGH containing the promoter GAP as described in the examples provided below and is designated Luck-α-IFN-1Y.

According to a further preferred embodiment, an expression vector is provided which is prepared by cloning the rhIFN-α gene into a vector comprising a promoter AG885 designed according to the invention as follows:

Alcohol dehydrogenase 2 (ADH2) of Saccharomyces cerevisiae is regulated by the presence of alcohol and glucose. It has been reported that when glucose in the medium is depleted, the expression of the ADH2 gene increases about 200-fold because the upstream activation sequence (UAS), which comprises 22 base pairs, is stimulated by the binding of the ADR1-activating protein to it (Yu et al., Molecular and Cellular Biology 9, 34-42 (1989)). Based on the above, the inventors of the present application have developed a novel promoter for gene expression in yeast comprising 200 base pairs of polynucleotide having the DNA sequence substantially identical to the TATA box of GAPDH and 90 base pairs of polynucleotide having the DNA sequence substantially identical to the upstream activation sequence of ADH2; and designated as promoter AG885.

The nucleotide sequence of the AG885 promoter is shown in Fig. 2.

The expression vector of the rhIFN-α gene inserting the AG885 promoter is prepared as described in the Examples below and designated pYLBC-AG885-α-IFN.

The expression vector of the invention, in particular pYLBC-AG885-α-IFN, gives a high yield of hIFN-α and at least about 10% of the total yeast protein.

Saccharomyces cerevisiae transformed with the vector Luck-α-IFN-1Y was deposited with the Korean Culture Center of Microorganism on December 12, 1990 with the deposit number KFCC 10715, and the conversion of the original deposit into a deposit under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure was requested on December 12, 1992, and the corresponding deposit number is KCCM 10019. Saccharomyces cerevisiae transformed with the vector pYLBC- AG885-α-IFN was deposited with the Korean Collection for Type Culture on December 17, 1991 with the deposit number KCTC 0028BP under the terms of the Budapest Treaty.

A yeast cell transformed with the expression vector containing the rhIFN-α gene is then cultured under conditions suitable for the expression of the rhIFN-α gene and determined according to the characteristics of the vector and the host yeast cell.

The hIFN-α produced in the transformed yeast can be purified by a combined application of conventional methods, e.g. cell disruption, centrifugation, dialysis, salting out, chromatography, gel filtration, electrophoresis and electroelution; then, hIFN-α having the same biological activity as the natural product is obtained in a high yield by the following procedure: dissolving the hIFN-α isolated from yeast cells in a solution containing a reducing agent; refolding the reduced hIFN-α under refolding conditions; subjecting the solution containing the refolded hIFN-α to anion exchange chromatography to elute the refolded hIFN-α as an eluate; subjecting the eluate to cation exchange chromatography to separate inactive hIFN-α from the active hIFN-α; and treating the inactive hIFN-α with a redox agent containing oxidized thiol residues and reduced thiol residues to convert it into active hIFN-α.

The inactive form of hIFN-α can be converted to active hIFN-α by treatment with a redox agent, particularly a redox agent containing oxidized thiol residues and reduced thiol residues in an appropriate ratio.

In the redox agent, the concentration of reduced thiol residues is preferably about 1 to 10 mM, and the concentration of oxidized thiol residues is preferably about 0.1 to 1 mM. The redox agent includes, for example, agents comprising cysteine and cystine, and also those comprising oxidized glutathione and reduced glutathione.

According to the above purification method, the polypeptide biologically identical to human alpha interferon can be purified in a high yield.

The following examples are intended to specifically illustrate the present invention without limiting the scope thereof; and the experimental procedures employed in the examples are carried out in accordance with the reference examples provided below.

Unless otherwise stated, percentages given below for solids in solid mixtures, liquids in liquids and solids in liquids are expressed on a w/w, v/v and w/v basis respectively.

Reference Example 1: Digestion of DNA with Restriction Endonuclease

The restriction endonucleases and reaction buffers used here were purchased from NEB (New England Biolabs, USA).

The reaction was generally carried out in a sterilized Eppendorf tube with a reaction volume in the range of 50 to 100 µl at a temperature of 37ºC for 1 to 2 hours. The reaction mixture was then heat treated at 65ºC for 15 minutes (or, in the case of a heat-stable endonuclease, phenol extracted and ethanol precipitated) to inactivate the restriction endonuclease.

10 · Reaction buffer for the reaction of a restriction endonuclease had the following composition:

10 · NEB reaction buffer 1: 100 mM bis-tris-propane-HCl, 100 mM MgCl₂, 10 mM dithiothreitol (DTT), pH: 7.0;

10x NEB reaction buffer 2: 100 mM Tris-HCl, 100 mM MgCl2 , 500 mM NaCl, 10 mM DTT, pH 7.0;

10 · NEB reaction buffer 3: 100 mM Tris-HCl, 100 mM MgCl₂, 10 mM DTT, pH 7.0; and

10 · NEB reaction buffer 4: 200 mM Tris acetate, 100 mM magnesium acetate, 500 mM potassium acetate, 10 mM DTT, pH 7.0.

Reference Example 2: Phenol extraction and ethanol precipitation

After completion of the enzyme reaction, the reaction mixture was phenol extracted to inactivate the enzyme or to recover DNA from the reaction mixture using phenol pre-equilibrated with a buffer containing 10 mM Tris-HCl (pH: 8.0) and 1 mM EDTA. Phenol extraction was performed by mixing equal volumes of a sample and phenol with vigorous shaking; centrifuging the mixture at 15,000 rpm for 5 minutes; and transferring the aqueous layer to a new tube. The above procedure was repeated 2 or 3 times.

The aqueous layer was then extracted with an equal volume of chloroform solution (chloroform:isoamyl alcohol = 24:1), and the aqueous layer was separated again; 0.1 volume of 3 M sodium acetate and 2.5 volumes of ethanol were added; and the mixture was centrifuged at 15,000 rpm and 4°C for 20 minutes after being kept at -70°C for 30 minutes or at -20°C for 12 hours to recover the nucleic acid.

Reference Example 3: Ligation reaction

Ligation of DNA was performed using T4 DNA ligase and 10 x ligation reaction buffer (0.5 M Tris-HCl, 0.1 M MgCl2, 0.2 M DTT, 10 mM ATP, 0.5 mg/ml bovine serum albumin (BSA)) purchased from NEB. The reaction volume was generally 20 µl, and 10 units of T4 ligase were used for the ligation of cohesive ends of DNA, while 100 units of T4 ligase were used for the ligation of blunt-ended DNA.

The reaction was carried out at 16°C for 5 hours or at 4°C for 14 hours; and after the reaction was completed, the reaction mixture was heated at 65°C for 15 minutes to inactivate the T4 ligase.

Reference Example 4: Transformation of E. coli

E. coli strains used for the following examples include E. coli HB101 (ATCC 33694), E. coli W3110 (ATCC 27325), and E. coli JM105 (ATCC 47016).

The host cells were incubated in 100 ml of liquid LB medium (1% Bacto-tryptone, 0.5% Bacto-yeast extract, 0.5% NaCl) at 37°C until the OD (optical density) at 650 nm reached a range of 0.25 to 5.0. The cell culture was isolated by centrifugation and then washed with 50 ml of 0.1 M magnesium chloride (MgCl2) solution. The cell pellet obtained by centrifugation was added with 50 ml of a solution of 0.1 M CaCl2 and 0.05 M MgCl2 and then left in an ice bath for 30 minutes. The cells were isolated by centrifugation and then suspended in 5 ml of a solution of 0.1 M CaCl2 and 0.05 M MgCl2. The reagents and materials used above were cooled to 0ºC immediately before use.

To 0.2 ml of cell suspension obtained above was added 10 µl of a ligation reaction mixture; and the reaction mixture was allowed to stand at 0°C for 40 minutes and then heated to 42°C for 1 minute. 2.0 ml of liquid LB medium was added; and the thus treated cells were incubated at 37°C for 1 hour. The culture was then transferred to a Petri dish containing solid LB medium supplemented with an appropriate antibiotic such as ampicillin and incubated at 37ºC overnight to obtain transformed colonies.

In the case of transformation of E. coli with the M13 vector, M13 vector DNA (ligated to a gene) was added to 100 μl of JM105 cell suspension, 15 μl of 10 mM isopropyl-β-thiogalactopyranoside, 25 μl of 4% X-Gal and 3 mL of 2× YT soft solid medium (1% NaCl, 1% yeast extract, 1.6% Bacto-tryptone, 0.7% Bacto-agar) prewarmed to 38°C. The mixture was stirred and applied to 2× YT solid medium (1% NaCl, 1% yeast extract, 1.6% Bacto-tryptone, 1.5% Bacto-agar) and incubated at 37°C overnight to obtain plaques.

Reference Example 5: Synthesis of oligonucleotides

Oligonucleotides were synthesized using a DNA synthesizer (Applied Biosystems Inc., 380B, USA) with automated solid-phase phosphoamidite chemistry.

The synthesized oligonucleotides were purified using denaturing polyacrylamide gel electrophoresis (2 M urea, 12% acrylamide and bisacrylamide (29:1), 50 mM Tris, 50 mM boric acid, 1 mM EDTA-Na2) and SEP-PAK column chromatography (Waters Inc., USA) using a mixture of acetonitrile and water (50:50) as eluent; and the amount was determined by measuring the OD value at 260 nm.

Reference Example 6: Polymerase Chain Reaction (PCR)

To a mixture of 10 to 100 mg of template DNA, 10 µl of 10 x Taq polymerase reaction buffer (10 mM Tris-HCl, 500 mM KCl, 15 mM MgCl2, 0.1% (w/v) gelatin, pH: 8.3), 10 µl of a mixture of dNTP (dGTP, dATP, dTTP and dCTP, each 1.25 mM), 2 µg of each primer (generally, 2 primers were used for the reaction, and in the case where 3 primers were used, the primer located in the middle was used in an amount of 0.02 µg) and 0.5 µl of Ampli-Taq DNA polymerase (Perkin Elmer Cetus, USA) was added distilled water in such an amount that the total volume was 100 μl; and 50 μl of mineral oil was added to protect the reaction mixture from evaporation.

PCR was performed using a thermal cycler (Perkin Elmer Cetus, USA); and the thermal cycle was programmed to be repeated 25 times or more, where the cycle was: 95ºC for 1 minute → 55ºC for 1 minute → 72ºC for 2 minutes; and finally the reaction was carried out at 72ºC for 10 minutes.

After completion of the reaction, the mixture was extracted with phenol, and the PCR products were recovered by precipitation with ethanol; and the precipitate was dissolved in 20 μl of TE buffer solution (10 mM Tris-HCl, 1 mM EDTA, pH: 7.5).

Example 1: Preparation of human alpha interferon cDNA (1-A): Production of mRNA from human cells < Step 1> : Extraction of total RNA from human cell line KG-1

The human cell line KG-1 (ATCC CCL246) was incubated in RPMI164 medium (Gibco, cat. #430-3400EB, Grand Island, New York 14072, USA) for 2 to 3 days; the medium was removed by centrifugation (750 x g, 10 min). The cell pellet (approximately 5 x 10⁷ cells) was suspended in fresh RPMI164 medium at a concentration of approximately 10⁷ cells/ml. To the suspension was added 1/200 volume of 200 mM sodium butyrate was added and the mixture was then incubated at 37ºC for 48 hours. The culture was centrifuged again and the cell pellet was isolated and suspended in fresh RPMI164 medium.

To induce the production of hIFN-α mRNA in the cells thus obtained, NCD virus (New Castle Disease virus, ATCC VR0107) was added to the cell suspension, and the mixture was then incubated at 37°C for 12 to 20 hours. About 5 x 107 cells induced as above were precipitated by centrifugation (750 x g, 10 min), and the cell pellet was lysed in 10 ml of RNAzol B (Cinna/Biotecs, USA). To the cell lysate, 1 ml of chloroform was added, and the mixture was shaken vigorously for 15 seconds and left in an ice bath for 5 minutes. The mixture was centrifuged at 12,000 x g, 4°C for 15 minutes to separate the water/chloroform phases. The upper phase was removed and treated with an equal volume of isopropanol.

The mixture was left at 4°C for 15 minutes and centrifuged at 1200 x g, 4°C for 15 minutes to precipitate RNA. The RNA precipitate was suspended in 200 μl of distilled water treated with diethyl pyrocarbonate (DEPC), and mRNA was tagged with a poly(A)+ (poly(A+)-mRNA) and immediately isolated therefrom.

< Step 2> : Isolation of poly(A)⁺ mRNA

Isolation of poly(A)+ mRNA was performed using an mRNA isolation kit (Stratagene Inc., Cat. #200348, USA) as follows.

The sample (200 μl) obtained in <Step 1> was heated at 65°C for 5 minutes and then 20 μl of 10 x sample buffer (10 mM Tris-HCl, 1 mM EDTA, 5.0 M NaCl, pH 7.5) was added on ice. The mixture was then immediately loaded onto an oligo-d(T) cellulose column at a rate of 1 drop/2 seconds. The oligo-d(T) cellulose column was then washed twice by loading each with 200 μl of high salt buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.5 M NaCl) at a rate of 1 drop/2 seconds. Poly(A)+ mRNA was eluted with 200 μl of low-salt buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.1 M NaCl). The eluted sample was subjected to ethanol precipitation and then dissolved in 100 μl of TE solution.

(1-B): Preparation of a cDNA library < Step 1> : Preparation of cDNA

For the preparation of cDNA from the poly(A)+ mRNA, the Zap cDNA synthesis kit (Stratagene Inc., USA, cat. #200400) was used. The poly(A)+ mRNA was used as a template, and an oligo d(T) primer with the nucleotide sequence 5'- GAGAGAGAGAGAGAGAGAGAGAACTAGTCTCG AG(T)18-3' was used.

The first strand of cDNA was prepared as follows. 18 µl of the mRNA solution prepared in Example (1-A) was mixed with 2 µl of 0.1 M CH₃HgOH, and the mixture was allowed to stand at room temperature for 10 minutes to unfold the secondary structure of RNA. To the resulting mixture was added 2 µl of 1 M β-mercaptoethanol, and the mixture was kept at room temperature for 5 minutes. To this were added 5 µl of reverse transcriptase reaction buffer solution (500 mM Tris-HCl, pH: 8.3, 750 mM KCl, 30 mM MgCl₂, 10 mM DTT), 2.5 µl of 10 mM each of dATP, dGTP, dTTP and 5- Methyl-dCTP, 2 μl of oligo d(T) primer (1.4 μg/μl), 15 μl of distilled water treated with DEPC, and 1.0 μl of RNase inhibitor (1 unit/μl, Promega Inc., USA) were added in the above order; the mixture was left at room temperature for 10 minutes to bind the primers to the template; then 2.5 μl of Superscript RNase H- reverse transcriptase (180 units/μl, BRL Inc., Cat. No. 8853SA) was added. The reaction mixture was incubated for 1 hour at 37°C to synthesize the first strand of cDNA.

The second strand of cDNA was prepared as follows. To 45 µl of the first strand solution obtained above, 40 µl of 10 x second strand buffer solution (188 mM Tris-HCl, pH: 6.9, 906 mM KCl, 46 mM MgCl2, 1.5 mM β-NAD (nicotinamide adenine dinucleotide), 10 mM (NH4)2SO4), 6.0 µl of 10 mM each of dATP, dCTP, dTTP and dGTP and 298 µl of distilled water were added in the above order; 1.0 μl of RNase H (4 units/μl) and 10.0 μl of DNA polymerase I (11 units/μl) were then added dropwise along the wall of the test tube. After immediate mixing, the reaction mixture was then incubated at 16ºC for 2.5 hours.

The resulting mixture was then extracted with an equal volume of phenol-chloroform mixture (1:1 (v/v), the phenol already saturated with 0.5 M Tris-HCl (pH 7.5) and 0.1% (v/v) β-mercaptoethanol) 3 times. The upper aqueous phase was removed and mixed with 0.1 volume of 3 M sodium acetate and 2 volumes of 100% ethanol. The mixture was left at -20°C overnight and centrifuged at 12,000 x g, 4°C for 20 minutes to obtain the cDNA precipitate.

< Step 2> : Preparation of a cDNA library

To convert double-stranded cDNA into blunt-ended, the cDNA precipitate prepared in the above <Step 1> was dissolved in 43.5 µl of distilled water. 39 µl of the cDNA solution was taken out and then mixed with 5.0 µl of T4 DNA polymerase reaction solution (670 mM Tris-HCl, pH: 8.8, 166 mM (NH₄)₂SO₄, 67 mM MgCl₂, 100 mM β-mercaptoethanol, 67 µM EDTA), 2.5 µl of 2.5 mM dNTP mixture and 3.5 µl of T4 DNA polymerase (2.9 units/µl). The reaction mixture was allowed to stand at 37°C for 30 minutes, and the product was extracted with the phenol-chloroform mixture and precipitated with ethanol in the same manner as in the above <Step 1>.

To provide recognition sites for restriction endonucleases EcoRI and XhoI at the 5'-end and 3'-end, respectively, the blunt-ended double-stranded cDNA prepared above was treated as follows: to the precipitate containing the blunt-ended cDNA obtained above were added 7.0 µl of EcoRI adapter (Stratagene Inc., Zap cDNA synthesis kit, Cat. No. 200400, USA), 1.0 µl of 10 X ligation buffer solution (500 mM Tris-HCl, pH: 7.8, 100 mM MgCl2, 200 mM DTT, 500 µg/ml BSA), 1.0 µl of T4 DNA ligase (1000 units/µl) and 1.0 µl of 10 mM ATP. The resulting mixture was left overnight at 4ºC and then heated to 70ºC for 10 minutes to inactivate the ligase.

The product thus obtained was extracted with the phenol-chloroform mixture and precipitated with ethanol in the same manner as above and then dissolved in 16 μl of TE buffer solution.

To the resulting solution, 2 µl of 10 x buffer solution (0.5 M NaCl, 0.5 M Tris-HCl, 50 mM MgCl2, 5 mM DTT, pH: 7.9) and 1 µl each of EcoRI and XhoI (New England Biolabs Inc., USA) were added, and the reaction mixture was then left at 37°C for 10 minutes to partially digest the cDNA. The cDNA fragments were extracted with the phenol-chloroform mixture and precipitated with ethanol in the same manner as above, and then dissolved in 10 µl of TE buffer solution.

The cDNA fragment thus obtained was cloned into the vector UNI-ZAPXR as follows. To 10 μl of the cDNA fragment solution digested with EcoRI-XhoI were added 2.0 μl of 10 x ligation buffer solution, 2.0 μl of 10 mM ATP, 4.0 μl of Vector UNI-ZAPXR solution (1 μg/μl, Stratagene Inc., USA) already digested with EcoRI/XhoI, and 2.0 μl of T4 DNA ligase (4 Weiss units/μl), and the reaction mixture was then incubated at 16°C for 10 hours.

<Step 3> : In vitro packaging of the vector-containing cDNA into phages and amplification of the cDNA library

To package the ligated DNA into a phage, 10 µl of the solution finally obtained in the above <Step 2> was added to Gigapack II Gold Packaging Extract (Stratagene Inc., USA), and the reaction mixture was left to stand at room temperature for 2 hours. To the resulting mixture were added 500 µl of a phage diluent solution (5.8 g NaCl, 2.0 g MgSO4·7H2O, 50 ml 1 M Tris-HCl, pH: 7.5, 5 ml 2% gelatin per liter) and 20 µl chloroform (see Kretz et al., Nucl. Acid. Res. 17, 5409 (1989)).

Infection and amplification were carried out as follows. PLK-F' (Stratagene Inc., Zap-cDNA Synthesis Kit, Cat. No. 200400), E. coli merA-, merB- strain was cultured in LB medium until the OD at 600 nm (OD600) reached 0.5. The cultured cells were precipitated and then dissolved in 10 mM MgSO4 to adjust the OD600 to 1.0. 600 µl of the solution was mixed with 20 µl of packing mixture; the reaction mixture was then left at 37°C for 15 minutes to allow infection of E. coli by the packaged phage particles. To the resulting E. coli was added 6.5 ml of 0.7% NZY agar (7 g NZ-amines, 5 g NaCl, 2 g MgSO4·7H2O, 5 g yeast extract, 7 g Bacto-agar per liter) melted and kept at 48°C; the mixture was spread on a 150 mm diameter NZY agar plate (7 g NZ-amines, 5 g NaCl, 2 g MgSO4·7H2O, 5 g yeast extract, 16 g Bacto-agar per liter) and then incubated at 37°C for 5 to 8 hours to generate phage plaques. 10 ml of the phage diluent solution was poured onto the plate; and the plate was gently shaken at 4°C for 15 hours to dissolve the phages; the resulting mixture was centrifuged at 4000 x g to precipitate E. coli cells, which were then removed. To the cDNA library solution thus obtained, 0.3% volume of chloroform was added; the titer of the cDNA library was determined to be about 10¹⁰ to 10¹³ PFU (plaque forming units)/ml. 100% DMSO (dimethyl sulfoxide) was added to adjust the concentration to 7% (vol/vol); the cDNA library was stored at -70°C.

(1-C): Screening of the cDNA library by immunoassay and determination of the cDNA sequence

The cDNA library was screened using the immunoscreening method described by Huynh, TV et al., DNA Cloning Techniques: A Practical Approach (DM Glover, ed.), pp. 49-78, IRL Press, Oxford (1985), to select for phages containing INF-α cDNA.

The cDNA library solution prepared in Example (1-B) was diluted to 50,000 PFU per 150 mm diameter plate. The diluted cDNA library solution was mixed with 600 µl of E. coli XL-1 Blue (Stratagene Inc., USA, Zap cDNA Synthesis Kit Cat. No. 200400) culture (OD₆₀₀ = 0.5) prepared by the same method as in <Step 2> of Example (1-B), and 6.5 ml of 0.7% NYZ agar was added. Each mixture was applied to a 40 NZY agar plate and cultured at 37°C for 12 hours to form 2 × 106 phage plaques.

Subsequently, nitrocellulose filters with a diameter of 132 mm were immersed in 10 mM IPTG (isopropyl-β-D-thiogalactopyranoside) solution and then dried by blotting on Whatman 3MM filters. The individual filters were placed over the agar on the individual plates and incubated for 3.5 hours at 37ºC. The individual filters that had undergone the transfer with the phage plaques were then washed with 15 ml of washing solution (10 mM Tris-HCl, pH: 8.0, 150 mM NaCl, 0.05% Tween 20). 15 ml of blocking solution (1% bovine serum albumin, 20 mM Tris-HCl, pH: 7.5, 150 mM NaCl) was added to the filters; followed by incubation at room temperature for 1 hour with gentle shaking. Each filter was then washed 5 times with gentle shaking using 15 ml of TBST buffer solution (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% (v/v) Tween-20) for 5 minutes at room temperature. Filters were placed in 15 ml of a solution prepared by diluting 100-fold anti-IFN-α antibody (cat. #853569; Boehringer Mannheim, USA) with TBS buffer solution (20 mM Tris-HCl, pH 7.5, 150 mM NaCl) containing 1% (w/v) FBS (fetal bovine serum) for 1 hour at room temperature with gentle shaking; and then washed 5 times for 5 minutes each at room temperature with gentle shaking in TBST buffer solution. Each filter was placed in 15 ml of a solution prepared by 2000-fold dilution of biotinylated goat anti-mouse IgG and avidin-conjugated alkaline phosphatase (Pierce Inc., USA) with TBS buffer solution containing 1% (w/v) FBS for 1 hour at room temperature with gentle shaking; and then washed 5 times for 5 minutes each at room temperature with gentle shaking in 15 ml of TBST buffer solution. Each filter was then dried by blotting on a Whatman 3MM filter.

For the staining reaction, each filter was reacted in 15 ml of staining solution (100 mM Tris-HCl, pH: 9.5, 100 mM NaCl, 5 mM MgCl2, 5 mg nitroblue tetrazolium, 2.5 mg 5-bromo-4-chloro-3-indolyl phosphate) in a darkroom for 30 minutes at room temperature. The purple positive phage plaques expected to express the cDNA encoding IFN-α were confirmed visually. Each filter was washed 1 time with TBS buffer solution; and a staining stop solution (20 mM Tris-HCl, pH: 2.9, 1 mM EDTA) was added to stop the staining process. Each filter was dried at room temperature and then recorded on a Polaroid film.

Positive plaques were isolated and incubated for 1 to 2 hours at room temperature in 1 ml of Phage diluent solution (10 mM Tris-HCl, pH 7.5, 10 mM MgCl2). The above immunoscreening assay was repeated to obtain the colonies as single phage plaques.

Each phage plaque confirmed to contain the gene encoding hIFN-α was placed in a sterilized microcentrifuge tube containing 500 μl of SM buffer solution (5.8 g NaCl, 2.0 g MgSO4, 50 μl 1 M Tris-HCl, pH 7.5, 5 mL 2% gelatin per liter); 20 μl of chloroform was added; and then the contents of the tube were cultured with shaking for 1 to 2 hours at room temperature. 200 µl (>1 x 10⁵ phage particles) of the thus obtained solution, 1 µl of helper phage R408 (>1 x 10⁵ PFU/ml, Stratagene Inc., USA) and 20 µl of E. coli XL-1 Blue cell suspension (OD₆₀₀ = 1.0) were mixed, and then the mixture was incubated at 37°C for 15 minutes. To the resulting culture was added 5 ml of 2 x YT medium (10 g NaCl, 10 g yeast extract, 16 g Bacto-tryptone per liter), and it was cultured with shaking at 37°C for 3 hours and then heated at 70°C for 20 minutes. The resulting culture was diluted 100-fold; 200 µl of the diluted culture was mixed with 200 µl of E. coli XL1-Blue cells (OD₆₀₀ = 1.0). After incubation at 37°C for 1 hour, 100 µl of the resulting culture was plated on LB plates containing ampicillin (50 µg/ml) and incubated at 37°C for 10 hours to obtain pBluescript phagemid colonies containing double-stranded cDNA.

To prepare single-stranded DNA, the pBluescript colonies obtained above were incubated in an LB agar medium containing the antibiotic tetracycline (12.5 µg/ml) and screened to obtain positive colonies again; the single positive colonies thus obtained were incubated in tetracycline+ LB broth medium (2 to 3 ml) overnight. The culture was then incubated in 0.3 ml of super liquid medium (35 g of Bacto-tryptone, 20 g of yeast extract, 5 g of NaCl, adjusted to pH 7.5 with NaOH) and cultured with shaking at 37°C. The culture was infected with helper phage R408 and culture was carried out for 8 hours until the OD600 reached 0.3.

Isolation and purification of the recombinant phage nucleotides was carried out according to the method proposed by J. Sambrook et al. in Molecular Cloning, I, 2.73-2.81, Cold Spring Harbor, NY(1989).

The nucleotide sequences were determined using purified single-stranded recombinant pBluescript phagemid or double-stranded pBluescript phagemid as a template and using M13-20-mer, primer T7, primer KS, primer SK or primer T3 (Stratagene Inc., USA) according to the method of Sanger (Proc. Natl. Acad. Sci. 74, 5405(1977)), the sequence shown in Fig. 1B with the amino acid sequences deduced from the nucleotide sequence.

Example 2: Modification of the nucleotide sequence of hIFN-α cDNA using synthesized oligonucleotides

The following four oligonucleotides were designed and synthesized so that the expression of hIFN-α cDNA was optimized by substituting the codons at the 5'-end of hIFN-α cDNA with the preferred codons for yeast without changing the amino acid sequence, while removing the ability to form secondary mRNA structure, so that the expression efficiency of hIFN-α in yeast was increased at the transcriptional and translational levels.

PCR was carried out using the above oligonucleotides. In a tube A, 2 µg of IFN1 and 2 µg of IFN2 were added; and in a tube B, 2 µg of IFN3 and 2 µg of IFN4 were added. To each of the tubes, 100 ng of DNA containing hIFN-α cDNA obtained in Example (1-C), 10 µl of 10 x Taq polymerase buffer, 10 µl of 10 mM dNTP and 2.5 units of Taq polymerase were added; distilled water was added to adjust the total volume to 100 µl. After the first PCR was carried out according to the method in Reference Example 6, PCR products of about 250 bp were isolated from each of the tubes A and B (hereinafter referred to as "PCR product A and B") by precipitation with ethanol.

Thereafter, a second PCR was performed using PCR products A and B under the same conditions as above. Approximately 510 bp nucleic acid fragment was obtained and designated as IFN-Y.

Example 3: Cloning of the rhIFN-α gene into the vector M13mp18 and determination of the nucleotide sequence

To 10 µl (about 2 µg nucleic acid) of TE buffer solution containing IFN-Y obtained in Example 2, 3 µl of 10 x NEB buffer solution 3, 16 µl of distilled water and 1 µl of restriction nuclease SalI (20 units/µl) were added; the mixture was reacted at 37°C for 1 hour and subjected to electrophoresis on 5% polyacrylamide gel to isolate 510 bp digested fragment. The isolated fragment was purified with Elutip-D (Schleicher & Schuell, USA). The purified fragment was designated as IFN-L and dissolved in 10 µl of distilled water before use for ligation.

On the other hand, double-stranded M13mp18 DNA (New England Biolabs, USA) was digested with restriction endonucleases SmaI and SalI, and the fragment was isolated in a purified form. To the mixture of 2 µl of 10 x ligation buffer solution (660 mM Tris-HCl, pH: 7.6, 66 mM MgCl2, 100 mM DTT), 2 µl of 100 mM ATP, 2 µl (0.2 µg) of IFN-L, 1 µl (about 0.1 µg) of M13mp18 DNA digested with SmaI and SalI, and 12 µl of distilled water was added 1 µl of T4 DNA ligase; the resulting solution was reacted for 5 hours at 16°C. Subsequently, 10 μl of the ligation result was reacted with E. coli JM105 (ATCC 47016) according to the Hanahan method described in J. Mol. Biol. 116, 557 (1983) for the transformation of E. coli cells.

The thus treated E. coli cell mixture was added with 7.5 μl of 0.2 M IPTG, followed by mixing with 3 ml of 2 x YT soft agar medium (16 g Bacto-tryptone, 10 g yeast extract, 5 g NaCl, 6 g Bacto-agar per 1 L) and 25 μl of 4% X-gal solution. The resulting mixture was incubated for Min A- Solid medium (10.5 g K₂HPO₄, 4.5 g KH₂PO₄, 1 g (NH₄)₂SO₄, 0.5 g Na citrate, 12 g Bacto agar, 1 ml 20% MgSO₄, 0.5 ml 1% vitamin B and 10 ml glucose per 1 L) and incubated at 37ºC overnight.

A white plaque thus obtained was isolated and used to obtain a single-stranded nucleic acid according to the method proposed by J. Sambrook et al., in Molecular Cloning, Cold Spring Harbor, NY (1989). The nucleotide sequence of the recombinant hIFN-α gene was determined according to the dideoxy DNA sequencing method (F. Sanger et al., PNAS, 84, 4767 (1987)); the rhIFN-α gene having the nucleotide sequence shown in Fig. 1A was selected; M13mp18 containing the nucleotide sequence was designated as M13mp18-IFN-α.

Example 4: Preparation of an expression vector (4-A): Preparation of the vector Luck-α-IFN-1Y

About 2 µg of plasmid pYLBC-GAP-HGH (Korean Laid-Open Patent Publication 90-9975) was completely digested with restriction endonucleases PstI and SalI in NEB buffer solution 3; another 2 µg of the plasmid was completely digested with restriction endonucleases PstI and BstBI in NEB buffer solution 4. Each of the digested plasmids was passed through 0.7% agarose gel to isolate fragments of about 9.8 kb and 3.4 kb, which were designated as fragments PL and PB.

About 5 µg of vector M13mp18-IFN-α containing the rhIFN-α gene obtained in Example 3 was completely digested with restriction enzymes XbaI and SalI in NEB buffer solution 3 and subjected to phenol-chloroform extraction and ethanol precipitation. The precipitated nucleic acid (hereinafter referred to as IFN-X/L) was dissolved in 10 µl of TE solution.

The ligation was performed using the fragments obtained above and a synthetic linker

carried out.

In a tube, 200 ng of fragment IFN-X/L, 50 ng of the linker, 100 ng of fragment PL, 100 ng of fragment PB, 2 µl of 10 x ligation solution and 10 units of T4 DNA ligase were added; and distilled water was added to make a total volume of 20 µl. The mixture was incubated at 16°C for 12 hours. The expression vector thus obtained containing a rhIFN-α gene was named Luck-α-IFN-1Y. The above procedure is shown in Fig. 3A.

(4-B): Preparation of the expression vector pYLBC-AG885-α-IFN < Step 1> : Synthesis of primers and PCR

The UAS of promoter ADH2 (5'-TCTCCAACTTATAAGTTGGAGA-3'), the TATA box of promoter GAPDH and the 111 bp DNA fragment containing a nucleotide sequence at the 5' end of the hIFN-α gene were subjected to PCR using two primers ADHGAP and GAPIFN as follows.

The primer ADHGAP (5'-AGACGGATCCTCCTCTGCCGGAACACCGGGCATCTCCAACTTAT AATGTTGGAGAAATAAGAGAATTTCAGATTGAGAGAATGAAAAAAAAAACTGAAAAAAAAGGTTGAA AC-3') and the primer GAPIFN (5'-TTCTTCTAGAACCCAGGCTGTGAGTTTGAGGCAGATCACACA TGGTGTTTGTTTATGTGTG-3') were synthesized.

The primer ADHGAP comprises 114 bases; the 94 bases at the 5' end contain a recognition site for BamHI and the UAS of the promoter ADH2; the remainder comprises a nucleotide sequence suitable for linking to the promoter GAPDH (-198th base to -178th base of GAPDH).

The primer GAPIFN contains a recognition site for the restriction endonuclease XbaI, an initiation codon for the hIFN-α gene and a nucleotide sequence of the promoter GAPDH comprising 18 bases (from the -1st to the -18th; Holland et al., J. Biol. Chem. 254, 9839-9845 (1979); Bitter et al., Gene 32, 263-274 (1984)) at the 3' end.

The structures of the primers are as follows. Primer ADHGAP

Using the two primers and a plasmid pYLBC5 containing the promoter GAPDH (ATCC 74119, cf. Korean Patent Application 91-25882) as a template, PCR was performed as follows,

To a mixture of 10 ng to 100 ng of plasmid pYLBC5 DNA as template DNA, 10 μl of 10 x Taq polymerase buffer (10 mM Tris-HCl, pH 8.3, 500 mM KCl, 15 mM MgCl2, 0.1 (w/v) gelatin), 10 μl of dNTP mixture (1.25 mM dGTP, dATP, dTTP and dCTP), 2 μg of primer ADHGAP, 2 μg of primer GAPIFN and 0.5 μl of Ampli-Taq DNA polymerase (Perkin Elmer Cetus, USA) was added distilled water to make a total volume of 100 μl, and 50 μl of mineral oil was added to prevent evaporation. A first PCR was performed using a thermocycler (Perkin Eimer Cetus, USA), repeating the thermal cycle: 95ºC for 1 min → 55ºC for 1 min; 72ºC for 2 min. and finally 72ºC for 10 min 25 times.

To the reaction mixture, an equal volume of phenol/chloroform was added; and the mixture was centrifuged to remove polymerase. The supernatant was transferred to a new tube; and 1/10 volume of 3 M sodium acetate and 2.5 volumes of 100% ethanol were added. The mixture was centrifuged to obtain about 320 bp DNA fragment, which was identified as AG885. The nucleic acid was dissolved in 20 μl of TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA) for use in the next step.

< Step 2> : Digestion of plasmid pYLBC5

2 µg of plasmid pYLBC5 was digested to completion with restriction endonucleases PstI and SalI in NEB buffer solution 3; another 2 µg of plasmid pYLBC5 was also digested to completion in the same manner. Each of the digested plasmids was subjected to 0.7% agarose gel electrophoresis to isolate fragments of approximately 9.84 kb and 3.0 kb, which were designated as fragments PK and PS, respectively.

< Step 3> : Isolation of a rhIFN-α gene fragment

About 5 µg of vector M13mp18-IFN-α containing the rhIFN-α gene obtained in Example 3 was completely digested with restriction endonucleases XbaI and SalI in NEB buffer solution 3 and subjected to agarose gel electrophoresis to obtain a fragment of about 450 bp of the rhIFN-α gene, which was designated as IFN-XL.

About 2 µg of the fragment AG885 obtained in the above <Step I> was completely digested with restriction endonucleases XbaI and BamHI in NEB buffer solution 3 and subjected to phenol/chloroform extraction and ethanol precipitation. The precipitated nucleic acid segment (hereinafter referred to as AG885-X/B) was dissolved in 10 µl of TE solution.

< Step 4> : Preparation of plasmid pYLBC-AG885-α-IFN

Fragments obtained in <Step 2> and <Step 3> above were ligated as follows. In a tube were added 200 ng of fragment IFN-X/L, 200 ng of fragment AG885-X/B, 100 ng of each of fragments PK and PS, 2 µl of 10 x ligation buffer and 10 units of T4 DNA ligase; distilled water was added to adjust the total volume to 20 µl. The mixture was reacted at 16°C for 12 hours. The above procedure is shown in Fig. 3B.

Example 5: Transformation of yeast with the expression vector and expression of the rhIFN-α gene < 5-A> : Transformation of yeast with Luck-α-IFN-1Y and expression of the rhIFN-α gene

The recombinant plasmid Luck-α-IFN-Y obtained in Example (4-A) was extracted in bulk by alkaline lysis (Ish-Horowicz, D. et al., Nucl. Acid Res., 9, 2989 (1981)) for use in the following transformation procedure. Transformation of yeast with the plasmid was carried out using the methods described by Veges (Nature 275, 104 (1978)) and Hinnen (PNAS 75, 1929 (1978)).

1 ml of S. cerevisiae DCO4 (Yeast Genetic Stock Center, Department of Biophysics and Medical Physics, University of California, CA 94720, USA) cultured overnight was inoculated into 50 ml of fresh YEPD medium and cultured with shaking at 30ºC until the OD value at 650 nm reached 0.8 to 1.0. The culture was centrifuged at 5,000 rpm for 5 minutes to isolate the cells. The precipitated cells were suspended in 20 ml of water; and the suspension was centrifuged again. The cell precipitates were suspended in 20 ml of 1 M sorbitol solution; and the suspension was centrifuged again.

The precipitated cells were dissolved in 5 ml SP (1 M sorbitol, 50 mM sodium phosphate, pH: 7.5) and 5 μl 1 M DTT; 50 μl xymolase (10 mg/ml in 50% glycerol/50% sp) was added, followed by culturing with shaking at 150 rpm and 30°C for 30 minutes. After culturing, a small amount of the culture was occasionally taken out and treated with 1/10 volume of 0.1% SDS to examine the degree of spheroplast formation with an optical microscope. Cultivation was carried out (generally for 30 minutes) until at least about 90% of the yeast cells had been converted into spheroplasts. After completion of cultivation, the culture was centrifuged at 1500 rpm for 3 minutes with a Dynac centrifuge to remove the spheroplasts. The spheroplasts thus obtained were washed with 5 ml of 1 M sorbitol and 5 ml of STC (1 M sorbitol, 10 mM CaCl2, 10 mM Tris-HCl, pH 7.5) and dissolved in 1 ml of STC before use in transformation with Luck-α-IFN-1Y.

To a mixture of 12.5 µl of 5 to 10 µg plasmid nucleic acid and 12.5 µl of 2 x STC was added 50 µl of the spheroplasts, followed by culturing for 10 minutes at room temperature. To the culture was added 500 µl of PEG/TC solution (40% PEG-4000, 10 mM CaCl2, 10 mM Tris-HCl, pH: 7.5), which was left for 10 minutes. The culture was centrifuged for 13 minutes at 1500 rpm with a Dynac centrifuge to isolate spheroplasts. The precipitated spheroplasts were dissolved in 200 µl of 1 M sorbitol solution.

The solution containing the spheroplasts was mixed with 7 ml of regeneration agar (3 g Bacto-agar, 50 ml 2 M sorbitol, 0.67 g yeast nitrogen base without amino acids, 0.1 g mixture of amino acids without leucine, 0.4 ml 50% glucose, 45 ml water per 100 ml); the mixture was poured onto a leucine-deficient plate (182 g sorbitol, 20 g Bacto agar, 6.7 g yeast nitrogen base without amino acids (Difco, USA), 0.25 g Leu-supplement, 4 ml 5% threonine, 40 ml 50% glucose and 820 ml water) and cultured at 30°C for 3 to 5 days to obtain yeast colonies transformed with the recombinant plasmid Luck-IFN-α-1Y.

A transformed yeast colony was cultured in 3 ml of leucine-deficient medium (6.7 g yeast nitrogen base without amino acids, 0.25 g mixture of amino acids without leucine and 6% glucose per 1 L) for 24 hours at 30ºC, then the culture was inoculated into 100 ml YEPD medium (2% peptone, 1% yeast extract, 2% glucose) and cultured for 24 hours at 30ºC.

The final OD of the culture was about 25 at 650 nm. The amount of the culture corresponding to an OD of 10 was centrifuged and dissolved in 400 μl of a buffer (10 mM Tris-HCl, pH: 7.5, 1 mM EDTA, 2 mM PMSF, 8 M urea). An equal volume of 0.4 mm diameter glass beads was added to the solution, followed by vigorous stirring to break the cell walls. 10 μl of the yeast extract thus obtained was subjected to electrophoresis on 15% SDS-polyacrylamide gel according to the method described by Laemmli et al. in Nature 277, 680 (1970) and stained with Coomassie Brilliant Blue R250 to confirm the expression of the gene product. The result is shown in Fig. 4A.

In Fig. 4A, lanes 1 and 2 both describe the extract from yeast cells containing the expression vector Luck-α-IFN-1Y; lane 3 shows the extract from yeast cells not containing the expression vector; and lane 4 shows standard protein size markers representing (from top) molecular weights 43,000, 29,000, 18,400, 14,300, 6,500, and 3,800 daltons. <

5-B> : Transformation of yeast with the expression vector pYLBC-AG885-α-IFN and expression of the rhIFN-α gene

The recombinant plasmid pYLBC-AG885-α-IFN obtained in Example (4-B) was prepared in bulk by an alkaline hydrolysis method (Ish-Horowicz, D. et al., Nucl. Acid Res. 9, 2989 (1981)) for use in transformation.

The transformation procedure of Example (5-A) was repeated using the plasmid pYLBC-AG885-α-IFN instead of the plasmid Luck-α-IFN-1Y to obtain yeast colonies transformed with the recombinant plasmid pYLBC-AG885-α-IFN.

The transformed yeast was cultured in 3 ml of leucine-deficient medium (6.7 g yeast nitrogen base without amino acids, 0.25 g of a mixture of amino acids without leucine and 6% glucose per 1 L) at 30ºC for 24 hours; then the culture was inoculated into 100 ml YEPD medium (2% peptone, 1% yeast extract, 2% glucose) and cultured at 30ºC for 24 hours.

The final OD value of the culture was about 25 at 650 nm. The amount of the culture corresponding to an OD value of 10 was centrifuged and dissolved in 400 μl of a buffer (10 mM Tris-HCl, pH: 7.5, 1 mM EDTA, 2 mM PMSF, 8 M urea). To the solution was added an equal volume of 0.4 mm diameter glass beads, followed by vigorous stirring to break the cell walls. 10 μl of the yeast extract thus obtained was subjected to electrophoresis on 15% SDS-polyacrylamide gel according to the method described by Laemmli et al. in Nature 277, 680 (1970) and stained with Coomassie Brilliant Blue R250 to confirm the expression of the gene product. The result is shown in Fig. 4B.

In Figure 4B, lane 1 shows the extract of yeast cells not containing the expression vector pYLBC-AG885-α-IFN; lanes 2 and 3 show the extract of yeast cells containing the expression vector pYLBC-AG885-α-IFN after culturing for 4 hours; and lanes 4 and 5 show the extract of yeast cells containing the expression vector pYLBC-AG885-α-IFN after culturing for 48 hours; and lane 6 shows the standard protein size markers representing (from top) molecular weights of 43,000, 29,000, 18,400, 14,300, 6,500, and 3,500.

Example 6 (for demonstration purposes only; not claimed as such): Purification of hIFN-α produced in yeast (6-A): Breaking the culture of transformed yeast

One kg of yeast transformed with the recombinant plasmid Luck-α-IFN-1Y obtained in Example (5-B) in which hIFN-α was expressed was suspended in a buffer (20 mM Tris-HCl, 1 M urea, 1 mM EDTA, 1 mM PMSF, pH: 8.0) using a homogenizer (Beckman) and disrupted at a rate of 750 ml/min with a Bead Beater (Biospec Product, USA) packed with glass beads having a diameter of 0.5 to 0.75 mm. The solution containing the disrupted yeast was centrifuged for 50 minutes at 12,000 rpm and 4°C with a centrifuge (Beckman, JA-14 rotor). The supernatant was discarded and the precipitates were collected. The obtained precipitates were suspended in 2 L buffer (20 mM Tris-HCl, 1 M urea, 1 mM EDTA, 0.1% Triton X-100, pH: 8.0) using a homogenizer (Beckman); and the suspension was centrifuged for 50 minutes at 12,000 rpm using a centrifuge (Beckman, JA-14 rotor). centrifuged to isolate the precipitates. The collected precipitates were further suspended in 2 L of 1 M guanidine solution (guanidine HCl, Amresco) using a homogenizer; and the suspension was centrifuged at 12,000 rpm for 30 minutes, isolating about 600 g of the precipitates.

(6-B): Dissolving the protein precipitates in a guanidine solution

600 g of the precipitates obtained in Example (6-A) were dissolved in 1.8 L of 6 M guanidine solution (6 M guanidine-HCl, 20 mM Tris-HCl, 100 mM β-mercaptoethanol, pH: 8.0) at 4°C overnight with stirring. The solution was centrifuged with a centrifuge (Beckman, JA-14 rotor) at 12,000 rpm for 30 minutes at 4°C to remove the precipitate. About 2.1 L of the collected dark brown supernatant was diluted 4-fold with a buffer solution (20 mM Tris-HCl, 0.1 mM PMSF, pH: 8.0) and centrifuged to collect the supernatant. The supernatant was concentrated to a volume of 2 L using an ultrafiltration membrane, and then the concentrate was dialyzed three times against 50 L of a buffer solution (20 mM Tris-HCl, 0.12 M NaCl, 0.1 mM PMSF, pH: 8.0) at 4ºC.

(6-C): DEAE-cellulose column chromatography

The solution obtained in Example (6-B) was centrifuged with a centrifuge (Beckman, JA-14 rotor) at 12,000 rpm for 30 minutes at 4°C to remove the precipitates. The collected supernatant was loaded onto a DEAE cellulose resin column (Whatman) equilibrated with a buffer solution (20 mM Tris-HCl, 0.12 M NaCl, 0.1 mM PMSF, pH: 8.0) at a rate of 10 cm/hour. The protein solution that passed through the column without binding to the resin was dialyzed 3 times against 50 L of a buffer solution (50 mM sodium acetate, pH: 4.5).

The dialyzed protein solution was centrifuged for 30 minutes at 12,000 rpm and 4ºC using a centrifuge (Beckman, JA-14 rotor) to remove the precipitates.

(6-D): CM-Sephalose column chromatography

A mixture of 50 mM sodium acetate and the dialyzed protein solution (pH: 4.5) obtained in Example (4-C) was loaded onto a CM-Sepharose resin column equilibrated with a buffer solution (20 mM Tris-HCl, 0.12 M NaCl, 0.1 mM PMSF, pH: 8.0) at a rate of 15 cm/hour. The column was washed with the same buffer solution to elute the proteins that did not bind to the resin until the peak of the proteins dropped sufficiently. Then, elution of protein was carried out using a buffer solution (50 mM sodium acetate, 0.12 M sodium chloride, pH: 4.5). When the peak decreased, further elution was performed with a buffer solution (50 mM sodium acetate, 0.5 M sodium chloride, pH: 4.5). The result of the CM-Sepharose chromatography is shown in Fig. 5 as the OD value at 280 nm.

In Fig. 5, the dark bar (a) shows fractions containing a partially oxidized hIFN-α; the dark bar (b) shows fractions containing mainly active form of monomeric hIFN-α which is essentially identical to natural hIFN-α; and the dark bar (c) shows fractions containing mainly dimeric hIFN-α. Partially oxidized hIFN-α, active form of hIFN-α and dimeric hIFN-α were eluted in sequence. To completely remove partially oxidized hIFN-α and dimeric hIFN-α from hIFN-α, the solution of fractions corresponding to the bar (b) was subjected to CM-Sepharose column chromatography to yield a highly purified form of monomeric hIFN-α. was received.

If necessary, further purification can be carried out by gel permeation chromatography.

The protein solution obtained above was passed through an S-300 (Pharmacia) column equilibrated with a buffer solution (20 mM NH4 acetate, 0.12 M NaCl, pH 4.5) at a flow rate of 3 cm/hour. The eluted fractions were subjected to non-reducing SDS-PAGE, and the fractions containing active form of monomeric hIFN-α and containing substantially no oligomeric hIFN-α were collected. The collected protein solution was dialyzed 3 times against 20 L of PBS buffer solution.

(6-E): Conversion of hIFN-α in inactive form into active form

Dimeric hIFN-α obtained in Example (6-D) ((c) of Figure 5) and identified by non-reducing 15% SDS-PAGE was collected and dialyzed against 20 mM Tris-HCl (pH 8.0).

To the above dialyzed solution, cysteine (Sigma) and cystine (Sigma) were added at a concentration of 5 mM and 0.5 mM, respectively, followed by stirring and standing for 5 hours at 4°C or at room temperature; the solution was subjected to SDS-polyacrylamide gel electrophoresis to confirm that the dimer had been converted to the monomer; and then the sample was dialyzed against a buffer solution (50 mM sodium acetate, pH: 4.5). Fig. 6 shows results of non-reducing 15% SDS-PAGE for the dimeric hIFN-α before the above conversion (a) and the monomeric hIFN-α converted above (b).

The same result was also obtained using an oxidized glutathione and a reduced glutathione.

The dialyzed sample was subjected to CM-Sepharose column chromatography and gel permeation chromatography according to the same procedure as in Example (6-D) to obtain a highly purified active form of monomeric hIFN-α.

The partially oxidized hIFN-α obtained in Example (6-D) ((a) of Figure 5) was subjected to the same conversion steps as above to obtain an active form of hIFN-α.

(6-F): Determination of the bioactivity of hIFN-α

The bioactivity of purified hIFN-α was determined by measuring the anticytopathic activity of hIFN-α against virus as follows:

A suspension of male calf kidney cells (MDBK, 3.5 x 105 cells/ml) cultured in a T75 flask was dispensed into 96-well plates in an amount of 100 µl and cultured in a 5% CO2 incubator at 37°C for 18 to 24 hours. 100 µl of vesicular stomatitis virus (1 x 105 to 4 x 105 pfu/ml) was inoculated into the wells, washed with PBS buffer (pH 7.0) and cultured in a 5% CO2 incubator at 37°C for 18 to 24 hours to form a monolayer of cells. 100 µl of each of the hIFN-α samples obtained in Examples (6-D) and (6-E) was diluted 2-fold in a dilution series, added to each well and left in a 5% CO2 incubator at 37°C for 48 hours. Then, a crystal violet dye solution was added to each well and the OD value at 600 nm was measured.

The protein concentration of hIFN-α was determined by the Lowry method. A standard hIFN-α, Gxa-01-901-535, was obtained from the NIH of the USA, and the specific activity of the same was 2.0 x 108 IU/mg.

The result of the activity determination showed that the two monomeric hIFN-α obtained in Examples (6-D) and (6-E) had a specific activity of 1.8 x 10⁸ IU/mg or more. Accordingly, the hIFN-α converted from the dimeric hIFN-α and the partially oxidized hIFN-α was equivalent to the active form of the monomeric hIFN-α initially obtained by CM-Sepharose chromatography in terms of biological activity.

(6-G): Reversed phase HPLC

Reverse-phase HPLC was performed to confirm the identity between the hIFN-α obtained in Examples (6-D) and (6-E).

Two protein samples were passed through a C18 column (u Bondapak, Waters) equilibrated with 15% (v/v) acetonitrile (15% acetonitrile/0.05% trifluoroacetic acid/H2O) with a rapid linear gradient of 25% (v/v) acetonitrile over 5 minutes and with a gentle linear gradient of 56% (v/v) acetonitrile over 35 minutes. The result is shown in Fig. 7, in which two proteins were eluted at the same position. Accordingly, it can be concluded that the hIFN-α obtained by the two above methods are identical in terms of biochemistry.

While the invention has been described with respect to the specific embodiments set forth above, it is to be understood that various modifications and changes which will be apparent to those skilled in the art to which the invention belongs may be made and are also within the scope of the invention as defined by the following claims.

Claims (8)

1. Recombinant human alpha interferon gene having the following nucleotide sequence: 2. An expression vector comprising the recombinant human alpha interferon gene of claim 1, wherein the gene is located at a site capable of being expressed in a yeast cell. 3. The vector of claim 2 which is Luck-α-IFN-1Y (KCCM 10019). 4. The vector of claim 2 which is pYLBC-AG885-α-IFN (KCTC 0028BP). 5. A yeast cell transformed with the expression vector of claim 2. 6. Transformed yeast cell according to claim 5, which is Saccaromyces cerevisiae DC04 Luck-α-IFN-1Y. 7. Transformed yeast cell according to claim 5 which is Saccaromyces cerevisiae DC04 pYLBC-AG885-α-IFN. 8. A process for producing human alpha interferon comprising culturing the yeast cell of claim 5, 6 or 7.