A METHOD OF PRODUCING HUMAN GAMMA-INTERFERON-LIKE POLIPEPTIDE

Abstract: A human gamma-interferon-like polypeptide is pro­ duced by cultivating host cells such as Eschrichia coli transformed with a synthesized polydeoxyribonucleotide. The human gamma-interferon activity of the polypeptide is confirmed by a test using an anti-human-interferon antibody.

Description: This invention relates to the production of a human gamma-interferon-like polypeptide (hereinafter abbreviated to γ-IFN). The chemically synthesized DNA molecule as disclosed herein is characterized by the DNA sequence coding for a polypeptide substantially agreeing in the amino acid sequence and composition with human gamma-interferon and having the immunological or biological activity of human gamma-interferon. Interferon includes three classes, namely alpha, beta and gamma, of proteins reportedly capable of inducing an antiviral condition in cells also showing antitumor activity. It is known that γ-IFN can be produced by the genetic engineering technique using a gene prepared by reverse transcription of a messenger RNA in the presence of a reverse transcriptase (Nature, 295 , 503, 1982). However, this technique is disadvantageous, among others, in that, since the gene sequence is defined by the messenger RNA, it is impossible to select codons adequate to host cells. Chemical synthesis of the γ-IFN gene may presumably overcome the above disadvantage and also may lead to construction of a chimeric polypeptide through addition of arbitrary restriction enzyme cleavage sites. However, the synthesis of said gene is not easy since it is composed of several hundreds of base pairs. The present inventors endeavored to overcome this difficulty and succeeded in synthesizing a γ-IFN gene. Thus, they developed a method of producing γ-IFN by the genetic engineering technique using said gene. The present invention thus provides a method of producing human gamma-interferon-like polypeptide which comprises cultivating in a nutrient medium host cells transformed with a plasmid with a polydeoxyribonucleotide, which has a translation-initiating codon at the 5'-end followed by a nucleotide sequence consisting of and a subsequent nucleotide sequence coding for the termination of translation, inserted therein, and thereby causing formation of the polypeptide having the above amino acid sequence as a protein component in the culture broth. The invention also includes the above-mentioned polydeoxyribonucleotide, plasmids incorporating such a polydeoxyribonucleotide, and Escherichia coil strains transformed with such a plasmid. Referring to the drawings, Figs 1 to 5 and Figs. 8 to 11 are given by way of supplement to the description of the example. Thus, sequence of each of 62 oligodeoxyribonucleotides used for the synthesis of a synthetic γ-IFN gene, Fig.1-(2) shows the amino acid sequence of human γ-IFN and the nucleotide sequence of the γ-IFN gene, Figs. 2 and 3 illustrate the oligodeoxyribonucleotide synthesis reaction process, Fig. 4 shows the oligodeoxyribonucleotide ligation scheme, Fig. 5 illustrates the scheme of determining the base sequence of the γ-IFN gene synthesized, and Fig. 8 shows the process of construction of plasmid pGIF. Fig. 9 is the 14% polyacrylamide gel electrophoresis mapping for the polydeoxyribonucleotide blocks A. B. C. D. E and F synthesized and for the molecular weight marker M ( Hinf I digest of  x 174RF), Fig. 10 is the 8% polyacrylamide gel electrophoresis mapping for each of the block products [I] and [II] of ligation according to the scheme shown in Fig. 4, and for the molecular weight marker M ( Hpa II digest of  x 174RF), and Fig. 11 is the 6% polyacrylamide gel electrophoresis mapping for each of the above blocks [I] and [II], the synthetic γ-IFN gene and the molecular weight marker M ( Hpa II digest of pBR322 DNA). Fig. 6 is the restriction enzyme cleavage map for the synthetic γ-IFN gene and Fig. 7 is the restriction enzyme cleavage map for the plasmid pKO703, the numerical values in the parentheses representing the distances in number of base pairs from the Eco RI recognition site. Fig. 12 is a graphic representation of the molecular weight of each fraction obtained by gel filtration of the supernatant which in turn was obtained by centrifugation of disrupted Escherichia coli cells and which exhibited interferon activity. A preferred example of the above-mentioned polydeoxyribonucleotide is the one having a sequence coding for the amino acid sequence 1-146 as shown in Fig. 1-(2) and having a translation-initiating codon, such as ATG, at the 5'-end and a translation-terminating codon, such as TAA, at the 3'-end. Because ATG codes methionine, γ-IFN of present invention naturally comprises a peptide added with formyl-methionine or methionine at the 5'-end of the amino acid sequence 1-142 mentioned above, as shown in Fig. 1-(2). For the sake of convenience in gene manipulation, it is preferable to add a nucleotide sequence capable of being cleaved with a restriction enzyme to each of the translation-initiating and translation-terminating codon endings. In a preferred embodiment, an Eco RI-cleavable nucleotide sequence is added to the 5'-end and a Sal I-cleavable nucleotide sequence to the 3'-end. The above polydeoxyribonucleotide can be synthesized, for example, in the following manner. First, the 62 gene fractions (oligodeoxyribonucleotides) shown in Fig. 1-(1) are synthesized by the per se known modified phosphotriester method and solid phase method. Then, a complete γ-IFN gene [cf. Fig. 1-(2)] is constructed by ligation of these fragments using T4 DNA ligase. This synthetic gene contains restriction enzyme cleavage sites, as shown in Fig. 6. This is for the purpose of facillitating search of active site structures or construction of chimeric genes. In the next place, the above gene is inserted into a vector plasmid. The following is an example of the insertion procedure. First, a plasmid, pKO703, is constructed by inser ting the promoter region (99 base pairs) of the Escherichia coli lactose operon between the Eco RI and Cla I cleavage sites of pBR322. Then, a plasmid, pGIF, is constructed by inserting the synthetic gene shown in Fig. 1-(2) and having an Eco RI cleavage site on the 5'-end side and a SalI cleavage site on the 3'-end side between the Eco RI and Sal I cleavage sites of the plasmid pKO703 (cf. Fig. 8). As the host cells, there may be used bacteria, yeasts and other fungi as well as cultured human and other animal cells and cultured plant cells. The bacteria are, for example, of the genus Escherichia, Bacillus or Pseudomonas. More specifical examples are Escherichia coli, Bacillus subtilis, Bacillus stearothermophilus, Pseudomonas putida and Pseudomonas aeruginosa. The yeasts are, for example, those belonging to the genus Saccharomyces. Saccharomyces cerevisiae is a preferred example. A strain belonging to the genus Escherichia is transformed with the plasmid pGIF obtained in the manner mentioned above. When Escherichia coli WA802 is used as said strain a transformant strain named WA802/pGIF is obtained. This strain has ampicillin resistance (Amp r ) and tetracycline sensitivity (Tet s ), owing to which it can be isolated from nontransformants. Upon inductive culture of the above transformant strain in a medium, such as L-broth, using isopropyl-B-D-thiogalactopyranoside, a substance having interferon activity is produced in the cells. As an example of a clone of the strain WA 802/pGIF, the strain Escherichia coil WA802/pGIF4 has been given a code name SBMG105 and deposited with the Fermentation Research Institute, Agency of Industrial Science and Technology, under Deposit No. FERM BP 282. A mode of practice of the present invention is described hereinbelow by way of example. Escherichia coli is sometimes referred to as E . coli . Example Chemical synthesis of gamma-IFN gene fragment The chemical synthesis of 62 oligodeoxyribonucleotides respectively designated F¹ through F₆₂ in Fig. 1-1, except for the improvements mentioned hereinafter, was carried out basically by the solid phase method reported by Ken-ichi Miyoshi et al [Nucl. Acids Res. 8 , 5507 (1980)]. The outline of chemical synthesis of these oligodeoxyribonucleotides by the improved technology is as follows. a) First, as shown in Fig. 2, the 3'-end mononucleoside unit was bound to an aminomethylated polystyrene resin. Thus, the aminomethylated polystyrene resin (1) shown in Fig. 2 is a resin cross-linked with 1% divinylbenzene, and the resin (2) was prepared by adding 2 units of beta-alanine to (1). Then, three different compounds (3) (B represents thymine, N-benzoylcytosine or N-isobutyrylguanine) were reacted with the above resin (2) to provide 3 different polystyrene resins (4) each carrying the 3'-end nucleoside. The following is an example of synthesis of the same resin (4) relevant to N-isobutyrylguanine for B in Fig. 2. Twenty grams of aminomethylated polystyrene·HCl (divinylbenzene 1%, 100-200 mesh, NH₂: 0.63 m mol/g) was swollen well with 100 ml of CH₂Cl₂ and N-t-BOC-beta-alanine (1.34 g, 15.1 m moles) was added. The condensation reaction was conducted in the presence of DCC (3.11 g, 15.1 m moles) at room temperature for 3 hours. The resin was filtered through a glass filter and washed twice with 200 ml portions of CH₂Cl₂, 3 times with 150 ml portions of DMF and finally 3 times with 100 ml portions of pyridine. Then, the resin was treated with 100 ml of 25% acetic anhydride-pyridine for 1 hour to acetylate the unreacted amino groups. Thereafter, the resin was treated with 120 ml of 20% CF₃COOH-CH₂Cl₂ at room temperature for 30 minutes to eliminate the t-BOC groups. The resin was washed with 200 ml of 5% triethylamine-CH₂Cl₂ and further washed 4 times with 100 ml portions of CS₂Cl₂. The above procedure was repeated for a second time to give the resin (2)of Fig. 2, i.e. the aminomethylated polystyrene resin having two β-alanine units. In 60 ml of DMF was suspended 10 g of the above resin [Fig. 2, (2)], followed by the addition of 11 m moles of compound (3) [Fig. 2, (3)] (where B means N-isobutyrylguanine) and 1 ml of triethylamine. The mixture was shaken at room temperature for 12 hours. After the reaction, the resin was separated by filtration, washed with DMF and, then, with pyridine, and finally treated with 150 ml of 10% phenyl isocyanate-pyridine for 3 hours to protect the unreacted amino groups. The resin was subsequently washed with pyridine-methanol and dried under reduced pressure in the presence of P₂O₅, whereby the resin (4) of Fig. 2 (B means N-isobutyrylguanine) was obtained. Using a portion of this resin (Fig. 2, (4), where B is N-isobutyrylguanine), the amount of 3'-end nucleoside secured to 1 gram of the resin was determined by the assay method of M.J. Gait et al [Nucl. Acids Res. 8 , 1081 (1980)]. The result was 0.12 m mole. The resin (4) wherein B is thymine or N-benzoylcytosine was also prepared in the same manner as above. The amount of 3'-end nucleoside secured to 1 g of resin (4) was 0.126 m mole for B-thymine and 0.125 m mol for B-N-benzoylcytosine. b) Using these three kinds of resin (4) and the 3'-phosphoric diester dimer block (Fig. 3, n=0) and 3'-phosphoric acid diester trimer block (Fig. 3, n=1), 62 oligodeoxyribonucleotides F₁ through F₆₂ having the specified predetermined base sequences. Thus, the resin (4) was treated with 10% trichloroacetic acid (briefly, TCA)-CHCl₃ to remove the dimethoxytrytyl group (briefly, DMTr) and, then, 4 equivalents of 3'-phosphoric acid diester dimer (Fig. 3, n=0) or 3 equivalents of 3'-phosphoric acid diester trimer (Fig. 3, n=1) per equivalent of the 3'-nucleoside unit secured to resin (5) was sequentially condensed with the aid of 20 to 50 equivalents of mesitylenesulfonyltetrazolide (MsTe). The excess 3'-phosphoric acid diester block and condensing agent could be easily removed by filtering the reaction mixture through a glass filter. Then, to protect the unreacted 5'-hydroxy group, the resin was either treated with 10% acetic anhydride-pyridine at room temperature for 1 hour or treated with a mixture of 10 ml of 10% acetic anhydride and 20 mg of 4-dimethylaminopyridine (DMAP) at room temperature for 30 minutes, whereby the unreacted 5'-hydroxy groups were acetylated. The resin was then treated with 10% TCA to remove the 5'-end DMTr group and, then, the next condensation reaction was conducted. The above procedure was repeated until the required length was obtained. An exemplary sequence of synthesis is given below with reference to the F₁₆ fragment. 200 mg of resin (4) [Fig. 2, (4), B=N-isobutyrylguanine] was treated 3 times with 20 ml of 10% TCA-CHCl₃ for 30 seconds each to give the resin (5) free from DMTr. To this resin (5) was added 96 µ moles of the 3'-phosphoric acid dimer block CA (Fig. 3, n=0, B₁=N-benzoylcytosine, B₃=N-benzoyladenine) and the azeotropic distillation with 3 ml of pyridine was repeated 3 times. Then, the resin was suspended in 2 ml of pyridine and the condensation reaction using 1 m mole of the condensing agent MsTe was carried out at room temperature for 2 hours. After the reaction, the excess 3'-phosphoric acid dimer and condensing agent could be easily removed by filtering the reaction mixture through a glass filter and washing the resin well with pyridine. Then, to protect the unreacted 5'-hydroxy groups, the resin was suspended in 20 ml of 10% acetic anhydride-pyridine and the suspension was shaken at room temperature for 1 hour. After this reaction, the resin was separated by filtration through a glass filter and washed well with pyridine and CHCl₃ in the order mentioned. Then, the resin was treated 3 times with 20 ml of 10% TCA-CHCl₃ for 30 seconds each, whereby the DMTr group was removed from the resin. The resin was washed well with CHCl₃ and pyridine in that order. The above procedure was repeated until the required length was obtained. Thus, to synthesize the oligodeoxyribonucleotide of F-16, the next 3'-phosphoric acid dimer blocks CC, TA, GA and TC were sequentially employed. By these sequential condensation reactions, a completely protected oligodeoxyribonucleotide-resin having the base sequence of F-16 was obtained. The other frangments F₁ to F₁₅ and F₁₇ to F₆₂ were also produced in the same manner except that different combinations of nucleotides were employed. c) The severence of the completely protected oligodeoxyribonucleotide having such a definite base sequence from the resin and the complete deprotection thereof were carried out by treating the resin with concentrated aqueous ammonia-pyridine (2:1, v/v) at 55°C for 10 hours and, then, with 80% acetic acid at room temperature for 10 minutes (Process A). However, the fragments F₂, F₃, F₅, F₇ to F₁₀, F₂₂ to F₂₄, F₂₆, F₂₉, F₃₀, F₃₃ to F₄₂, F₅₃ to F₅₅, and F₅₇ to F₆₂ could not be obtained under the above deprotection conditions. Therefore, these fragments were completely deprotected by the method of Reese et al. [Nucl. Acids Res. 9 , 4611 (1981)] (Process B). This deprotection procedure is described below with reference to F₁₆ and F₂₆ fragments. Process A: In a sealed tube, 50 mg of the F₁₆-resin was shaken with 1 ml of pyridine and 2 ml of concentrated aqueous ammonia at 55°C for 10 hours, at the end of which time the resin was filtered off and the filtrate was concentrated under reduced pressure. To the residue was added toluene and the azeotropic distillation was repeated to remove the pyridine. The residue was treated with 2 ml of 80% acetic acid at room temperature for 10 minutes, whereby a completely deprotected oligodeoxyribonucleotide was obtained. Process B: 50 mg of the F₂₆-resin was treated with a solution (0.3 M) of 1,1,4,4-tetramethylguanidium 2-pyridinealdoximate in 3.5 ml of dioxane-water (7:1, v/v) at 40°C for 48 hours and, then, in a sealed tube, it was further treated with 3 ml of concentrated aqueous ammonia-pyridine (2:1, v/v) at 55°C for 10 hours. Thereafter, the F₂₆-resin was treated with 2 ml of 80% acetic acid at room temperature for 20 minutes to give a completely deprotected oligodeoxyribonucleotide. d) The isolation and purification of the final product was carried out by high performance liquid chromatography (briefly, HPLC. Thus, ALTEX Model 110 A Liquid Chromatograph, a linear gradient method using Solvent A (20% CH₃CN-l m MKH₂PO₄, pH 6.5) and Solvent 9 (20% CH₃CN-500 mM KH₂PO₄, pH 6.5), and a Partisil 10 SAX (Whatman) column (4.6 dia. x 250 mm) were employed. The gradient was established by adding 3% of Solvent B to Solvent A at the intervals of 1 minute. Elution was carried out at 58°C and a flow rate of 2 ml/min. The fractions containing the desired compound were pooled, desalted by dialysis and lyophilized. The homogeniety of the 62 completely deprotected oligodeoxyribonucleotides F₁ through F₆₂ thus obtained was confirmed by labeling their 5'-ends with [gamma-³²P]ATP using T₄ polynucleotide kinase and carrying out polyacrylamide gel (20%) electrophoresis. The base sequence of each oligodeoxyribonucleotide was confirmed by the two-dimensional mapping method [Bambara, J.E. et al (1974) Nucl. Acids. Res. 1 , 331]. Thus, the 5'-end was labeled with[gamma-³²P]ATP using T₄ polynucleotide kinase and, then, partial hydrolysis was carried with nuclease. This partial hydrolysate was subjected to cellulose acetate electrophoresis (pH 3.5) and DEAE-cellulose homochromatography. Ligation of gamma-IFN gene fragments Using the 62 oligodeoxyribonucleotide fragments (1 to 62) synthesized in the above manner, the gene (Fig. 1-2) corresponding to γ-IFN consisting of 450 base pairs were ligated according to the ligation schema shown in Fig. 4. Details of the procedure are set forth below. [A] to [F] blocks were synthesized. Excepting the 5'-end fragments of each block (1, 14, 15, 24, 25, 32, 33, 44, 45, 54, 55, 62), 1 n mole of each of the remaining 50 fragments was dissolved in 22 µl of distilled water and the solution was heated at 90°C for 2 minutes and, then, immediately cooled to 0°C. To this aqueous solution was added 5 µCi of [γ-³²P] ATP (5100 Ci/m mole). It was then adjusted to give a 70 mM Tris-HCl (ph 7.5)-10 mM MgCl₂ kinase buffer solution. Then, 3 units of polynucleotide kinase was added to make a total of 30 µl. By conducting the reaction at 37°C for 15 minutes, the 5'-ends were labeled with ³²P. Then, to phosphorylate all the 5'-ends, 4 n moles of ATP and 3 units of polynucleotide kinase were further added and the reaction was continued at 37°C for 45 minutes. The reaction was terminated by heating the mixture at 90°C for 5 minutes. 2.1 µg each of the above phosphorylated fragments 2 through 13 and unphosphorylated fragments 1 and 14 (A Block), phosphorylated fragments 16 through 23 and unphosphorylated fragments 15 and 24 (B Block), phosphorylated fragments 26 through 31 and unphosphorylated fragments 25 and 32 (C Block), phosphorylated fragments 34 through 43 and unphos phorylated fragments 33 and 44 (D Block), phosphorylated fragments 46 through 53 and unphosphorylated fragments 45 and 54 (E Block), and phosphorylated fragments 56 through 61 and unphosphorylated fragments 55 and 62 (F Block) were respectively mixed and dialyzed in a dialysis tube against distilled water at 4°C for 16 hours to remove the unreacted ATP and kinase buffer components. This DNA dialyzate for each of A to F blocks was concentrated to dryness and dissolved in 14.5 µl of ligase buffer (20 mM Tris-HCl, pH 7.6, 10 mM MgCl₂). This solution was put in a 40 µl fine-gauge glass tube and after both ends of the tube were sealed, it was heated in a 16.5 mm test tube containing 20 ml of water at 95°C for 2 minutes. Then, the test tube as a whole was allowed to stand at room temperature for 2 hours to aneal the DNA fragment. It was then placed at 0°C. This DNA solution was taken out of the fine-gauge glass tube and made into a ligase buffer containing 0.4 mM of ATP and 10 mM of DTT. Then, 30 units of T4 DNA ligase was added to make a total volume of 80 µl and the reaction was allowed to proceed at 15°C for 16 hours. A portion of the reaction mixture was taken and subjected to electrophoresis with 14% polyacrylamide gel containing 7 M of urea and the state of ligation was investigated by radioautography. As a result, the largest ligation reaction products were found to agree in size with the desired products for 6 blocks A through F. Then, the entire amount of the above ligation product was fractionated by electrophoresis through 14% polyacrylamide gel containing 7 M of urea. After the positions of A through F were confirmed by radioautography, the gel portions corresponding to the positions were cut out and placed in dialysis tubes. Each tube was filled with 9mM Tris-borate buffer (pH 8.3, 0.25 mM EDTA added) (elution buffer) and after closing both ends of the tube, electrophoresis was conducted in the elution buffer at a constant voltage of 200-V for 3 hours to elute the DNA in the gel. The DNA was taken out from the dialysis tube and lyophilized. The lyophilisate was dissolved in 100 µl of 0.3 M sodium acetate (pH 7.0), and two volumes of ethanol were added. The mixture was allowed to stand at -80°C for 30 minutes, at the end of which time it was centrifuged to recover the DNA as a sediment. The DNA sediments for A through F blocks were dissolved in 20 µl of distilled water and a 1 µl portion of the solution was investigated by electrophoresis with 14% polyacrylamide gels containing 7 M of urea. As shown in Fig. 9, substantially uniform bands corresponding to the sizes of DNAs for the respective blocks [A] through [F] were obtained. The DNA solution for each of A through F blocks was adjusted to the composition of the kinase buffer, and 2 n moles of ATP and 6 units of polynucleotide kinase were added. This reaction was conducted at 37°C for 1 hour, whereby the 5'-ends of the blocks were phosphorylated. The DNA for [A], [B] and [C] blocks (20 p moles each) were mixed and ATP and DTT were added in the concentrations of 0.4 mM ATP and 10 mM DTT and to make a total of 50 µl. Then, using 8 units of T4 DNA ligase, the mixture was reacted at 15°C for 16 hours. The DNA's for [D], [E] and [F] blocks were also mixed in the proportions of 5 p moles each and the ligation reaction was conducted at 15°C for 16 hours. After the above reaction, a portion of each reaction product was taken and the state of ligation was examined by electrophoresis with 8% polyacrylamide gels containing 7 M of urea. As shown in Fig. 10, it was found that [I] and [II] blocks corresponding to 225 bp had been synthesized. Then, the entire amount of the above reaction mixture was fractionated by electrophoresis through 8% polyacrylamide gels containing 7 M of urea. After the position of the 225 base-pair DNA was confirmed by radioautography, the corresponding portion of the gel was cut out and the DNA was eluted by the above-mentioned electro phoretic elution method. The DNA solution was lyophilized and dissolved in 100 µl of 0.3 M sodium acetate (pH 7.0), followed by addition of 2.5 volumes of ethanol, whereby the DNA was precipitated. To this DNA were added 0.5 n mole of ATP and 3 units of polynucleotide kinase and the reaction conducted at 37°C for 1 hour in the same manner as described hereinbefore. The DNA's (1 p mole each) of blocks [I] and [II] were mixed and adjusted to the ligase buffer composition. Then, ATP and DTT were added to the mixture in the concentrations of 0.4 mM ATP and 10 mM DTT to make a total of 20 µl. Using 5 units of T4 DNA ligase, the ligation reaction was conducted at 15°C for 16 hours. After the reaction, a portion of the reaction mixture was taken and the state of ligation was investigated by electrophoresis with 6% polyacrylamide gel containing 7 M of urea. The result is shown in Fig. 11. In Fig. 11, 1 and 11 stand for the isolated and purified products for [I] and [II] blocks, respectively. Further, G denotes the result of ligation of the total γ-IFN gene, indicating that a DNA molecule corresponding to 450 base pairs was obtained. To the whole amount of the above reaction mixture was added 1/10 volume of 3 M sodium acetate, followed by addition of 2.5 volumes of ethanol, whereby the DNA was recovered as a precipitate. This precipitate was dissolved in 100 µl of TA buffer (33 mM Tris-acetate, pH 7.9, 66 mM potassium phosphate, 10 mM magnesium acetate, 0.5 mM DTT), and 20 units of restriction enzyme Eco RI and 20 units of restriction enzyme Sal I were added. The reaction was conducted at 37°C for 1 hour. To the reaction mixture was added 10 µl of 0:3 M sodium acetate and, then, 2.5 volumes of ethanol was further added, whereby the DNA was recovered as a precipitate. This DNA was dissolved in a solution containing 80% formamide, 10 mM NaOH and 1 mM EDTA and the solution was subjected to electrophoresis with 6% polyacrylamide gels containing 7 M of urea. After confirming 450 base pairs by radioautography, the corresponding gel was cut out and put in a dialysis tube. The tube was filled with the elution buffer and after sealing, electrophoresis was conducted in the same buffer at a constant voltage of 200 V for 3 hours, whereby the DNA in the gel was eluted. The DNA solution was taken out from the dialysis tube and lyophilized. The lyophilisate was dissolved in 100 µl of 0.3 M sodium acetate (pH 7.0) and 2.5 volumes of ethanol was added, whereby the DNA was recovered as a precipitate. By the above procedure was obtained about 0.05 p mole of γ-IFN gene. This DNA was dissolved in 10 µl of the kinase buffer and 2 units of polynucleotide kinase and 0.1 n mole of ATP were added. The reaction was conducted at 37°C for 1 hour to provide a DNA for transformation. Construction of pGIF plasmid For the expresion of the chemically synthesized gene as a complete protein in Escherichia coli , an Escherichia coli plasmid pKO 703 into which the γ-IFN gene can be inserted downstream of the UV5 promoter operator region of the Escherichia coli lactose operon was constructed, and the γ-IFN gene was cloned in this pKO703 plasmid. The details of construction of the series of plasmids employed in this experiment are set forth below (Fig. 8). (1) Construction of pKO703 plasmid The plasmid pKO703 containing the UV5 promoter-operator region of E . coli lactose operon was constructed by the following procedure. (A) Preparation of pBR 322 plasmid with the EcoRI and ClaI cleavage sites filled in pBR 322 DNA (1.5 µg) was cleaved at 37°C for 60 minutes with 5 units each of the restriction enzymes EcoRI and Cla I in 20 µl of 1 x TA mixture (containing 33 mM Tris-acetate buffer, pH 7.6, 66 mM potassium acetate, 10 mM magnesium acetate and 0.5 mM dithiothreitol). The reaction mixture was heated at 65°C for 30 minutes so as to inactivate the enzymes. Addition of 2.5 volumes of cold ethanol gave a DNA precipitate. The DNA precipitate was suspended in 40 µl of T4 DNA polymerase solution and heated at 65°C for 10 minutes, followed by addition of 5 µl each of dTTP, dATP, dCTP and dGTP from the respective 4 mM solutions and 5 µl of 100 mM β-mercaptoethanol. Then, the mixture was reacted with 1 unit of T4 DNA polymerase at 37°C for 60 minutes. The reaction mixture was heated at 65°C for 30 minutes to inactivate the enzyme and precipitated with ethanol. Finally, the DNA precipitate was dissolved in 15 µl of distilled water. (B) Preparation of the DNA in the Lac UV5 promoter- operator region with the EcoRI cleavage sites filled in To separate the UV5 promoter-operator region of lactose operon, the following experiment was conducted. Plasmid pKO601 is a plasmid having the Lactose operon promoter operator region between two Eco RI cleavage sites. 80 µg of this plasmid pKO601 was treated with 150 units of restriction enzyme Eco RI in 500 µl of 1 x TA mixture at 37°C for 60 minutes. The reaction mixture was subjected to 10% polyacrylamide gel electrophoresis. Thus, the DNA fragment of the promoter-operator region consisting of 95 base pairs and having Eco RI cohesive ends was separated by gel electrophoresis. (For details of the separation procedure, see Japanese Patent Application No. 163303/1981, Examples,the disclosure of which is republished in "Nucleic Acids Research 10(5), 1741, 1982.) After ethanol precipitation, the DNA precipitate was dissolved in 40 µl of T4 DNA polymerase solution and the solution was heated at 65°C for 30 minutes and, then, quenched. Thereafter, 5 µl each of dATP, DTTP, dCTP and dGTP from the respective 4 mM solutions as well as 5 µl of 100 mM B-mercaptoethanol was added. The mixture was reacted using 10 units of T4 DNA polymerase at 37°C for 60 minutes. The reaction mixture was heated at 65°C for 30 minutes to inactivate the enzyme and the DNA was precipitated with ethanol. Finally, the DNA precipitate was dissolved in 20 µl of distilled water. (C) Ligation of the plasmids prepared in (A) and (B) to the lactose UV5 promoter-operator region, and transformation thereof into Escherichia coli Using 0.8 µg of the pBR 322 DNA with Eco RI and Cla I cleavage sites filled in as prepared in (A), 0.8µg of the lactose operon UV5 promoter-operator region DNA as prepared in (B), and 2.5 units of T4 DNA ligase, the ligation reaction was conducted at 16°C for 20 hours. (This ligation reaction was conducted in accordnace with the Examples of Japanese Patent Application No. 163303/1981). After the ligation, the DNA was precipitated from ethanol and dissolved in 10 µl of distilled water. Using this DNA solution, transformation of Escherichia coli K12 WA802 was carried out. (This transformation was carried out in accordance with the method described in Japanese Patent Application No. 163303/1981.) The transformant was cultivated on a M9S-X-gal medium [ NaCl , 5 g; NH₄Cl , 1 g; Na₂HPO₄, 5.94 g, KH₂PO₄ , 3 g (pH 7.0), MgSO₄ , 0.2 g; casamino acid, 2 g; glucose, 5 g; 5-bromo-4-chloro-3-indoyl-β-D-galactopyranoside, 400 mg; agar,15 g; water, 1000 ml; hereinafter briefly, M9S-X-gal medium] containing 40 µg/ml of ampicillin, and 10 ampicillin-resistant, deep blue colonies were picked up and designated as WA802/pKO701∿WA802/pKO710. The recognition site of restriction enzyme Cla I on plasmid pBR 322 lies within the promoter region of the tetracycline-resistant gene and if another DNA fragment is inserted between Eco RI and Cla I recognition sites on pBR 322 plasmid, Escherichia coli having such plasmid becomes tetracycline-sensitive. However, it is expected that if the DNA fragment in the UV5 promoter-operator region of the lactose operon is inserted with the desired directionality, the transformant having the plasmid will become tetracycline-insensitive. Therefore, the WA802/pKO701 through WA802/pKO710 strains obtained above were cultivated by single colonization on the ordinary nutrient agar medium containing 10 µg/ml of tetracycline to examine their tetracycline sensitivity. It was found that WA 802/pKO701, WA802/pKO703, WA802/pKO706 and WA802/pKO709 were tetracycline-insensitive. Therefore, plasmid DNA was separated from each of these strains and 2 µg of DNA was reacted in 1 x TA mixture with 5 units of restriction enzymes Eco RI and Hpa II at 37°C for 60 minutes and the directionality of the lactose operon promoter-operator region was investigated by 10% polyacrylamide gel electrophoresis. It was found that in the case of WA 802/pKO703 and WA802/pKO709, the promoter-operation region had been inserted in the desired orientation. This pKO703 plasmid was used in the cloning of the γ-IFN gene. The plasmid pKO703 has Eco RI recognition site downstream in the transcription direction from the promoter, with the tetracycline-resistant gene of pBR 322 remaining intact on the plasmid, the gene having the cohesive ends of a restriction enzyme for cleaving the tetracycline-resistant structural gene, such as Eco RI, Sal I and BamHI obtained chemically or from a plasmid, can be easily introduced by ligation into the plasmid pKO703 and the desired clone can be easily selected as an ampicillin-resistant, tetracycline-sensitive strain. (2) Construction of plasmid pGIF₄ In 30 µl of 100 mM tris-HCl buffer (pH 7.3)-5 mM MgCl₂-50 mM Nacl, 15 µg of plasmid pKO703 was reacted with 10 units each of restriction enzymes Eco RI and Sal I at 37°C for 60 minutes. The reaction mixture was heated at 65°C for 30 minutes to inactivate the enzymes and, then, subjected to 0.7% agarose gel electrophoresis. By electrophoresis, 3.8 kb of DNA fragment was separated from the gel. After ethanol precipitation, the DNA was dissolved in 4.5 µl of distilled water. The resulting Eco RI/ Sal I cleaved fragment DNA (0.3 µg) and the 5'-OH end phosphorylated γ-IFN gene (24 µl) previously obtained were dissolved in T4 DNA ligase mixture and the solution was heated at 65°C for 10 minutes. After cooling with ice, 3 µl of 100 mM DTT and 3 µl of 4 mM ATP were added, and using 3 units of T4 DNA ligase, ligation was carried out at 16°C for 20 hours. After this ligation reaction, 2.5 volumes of cold ethanol was added, whereby the DNA was precipitated. To this precipitated DNA was added 10 µl of distilled water and the transformation of E. coli WA802 was conducted. The transformant was cultivated on an M9S-X-gal medium containing 40 µg/ml of ampicillin and ampicillin-resistant and deep blue colonies were picked and transferred to an ordinary nutrient agar medium containing 10 µg/ml of tetracycline by single colonization. Tetracycline-sensitive colonies were selected and designated as WA802/pGIF₁ through WA802/pGIF₅. After separation of plasmid DNA's, each plasmid DNA was cleaved with restriction enzymes Eco RI and Sal I, Eco RI and Hind III, or Eco RI and Bgl II and the sizes of DNA fragments were measured by agarose gel electrophoresis and polyacrylamide gel electrophoresis, In this manner, WA802/pGIF₄ and WA802/pGIF₅ wherein the desired γ-IFN gene had been cloned were confirmed. In these plasmids pGIF₄ and pGIF₅, the start codon ATG of the γ-IFN gene is situated 10 base pairs downstream of the Shine-Dalgano DNA base sequence in the UV₅ promoter region of the lactose operon. Identification of the base sequence of a γ-IFN gene The base sequence in the region corresponding to the 450 base pair γ-IFN gene obtainable by the Eco RI/ Sal I cleavage of the pGIF₄ plasmid DNA was identified according to the schema shown in Fig. 5. The determination of the base sequence was carried out in accordance with the Maxam-Gilbert method (Proc. Natl. Acad. Sci., U.S.A., 74 , 560-564, 1977). It was confirmed that throughout all the 450 base pairs the sequence was as initially designed (Fig. 1-2). (3) Expression and identification of a human gamma-interferon-lake polypeptide in the E . coli cell In E. coli WA802/pGIF₄ and WA802/pGIF₅, the Y-IFN gene was inserted downstream of the promoter region of the lactose operon. Therefore, it was expected that the γ-IFN would be expressed under the control of the lactose operon. Accordingly, the expression and fixation of γ-IFN in the cells of E . coli WA802/pGIF₄ and WA802/pGIF₅ were carried out. Each of E . coli WA802/pGIF₄ and WA802/pGIF₅ strains was used to inoculate 2.0 ml of M9S-glycerol medium (NaCl, 5 g; NH₄Cl, 1 g; Na₂HPO₄, 5.94 g; KH₂PO₄, 3 g; MgSO₄·7H₂O , 0.2 g; glycerol, 2.5 g, casamino acid, 2 g; water, 1000 ml) containing 40 µg/ml of ampicillin and the inoculated medium was incubated at 37°C overnight. The resulting culture was transferred to 4 tubes of M9S-glycerol medium (5 ml) containing 40 µg/ml of ampicillin in a size giving an absorbance (OD 660 nm) of 0.1. Incubation was carried out at 37°C and when the OD 660 nm reached 0.8, isopopyl-β-D-thiogalactopyranoside (briefly, IPTG) as a lactose operon inducer was added to two of the 4 tubes at the final concentration of 1 mM, further followed by incubation at 37°C for 2.5 hours. The remaining two tubes were incubated without IPTG at 37°C for 2.5 hours. A 5 ml portion each of these cultures was centrifuged at 3,000 r.p.m. for 10 minutes, whereby cells were recovered as a sediment. Then, 0.5 ml of 1 x PBS (10 mM phosphate buffer pH 7.0, 150 mM NaCl) containing 1 mg/ml of bovine serum albumin was added to each of the two tubes, one containing IPTG and the other not containing IPTG and the cells were distrupted by ultrasonication. To each of the remaining 2 tubes, one containing IPTG and the other not containing IPTG, was added 0.5 ml of 1 x PBS containing 1 mg/ml of egg white lysozyme. The lysozyme treatment was conducted at 0°C for 40 minutes, followed by ultra-sonication. 0.5 ml of the disrupted cells were centrifuged at 25,000 r.p.m. for 30 minutes at 4°C and the supernatant was used for the activity assay of γ-IFN. Determination of interferon activity Using a Coaster's 96-well plastic multiplate and a Eagle's minimal medium containing 5% of fetal calf serum, a dilution series of interferon samples were prepared by multi-pipetting. Then, 100 µl of FL cells (5 x 10⁵ cells/ml) were added to each well and the cultivation was conducted at 37°C overnight in a 5% CO₂ gas atmosphere. The culture supernatant was discarded and 100 µl of Sindbis virus in a concentration 100 times as high as TCID₅₀ was added to each well and the incubation was carried out at 37°C overnight in a 5% CO₂ gas atmosphere. After 17 hours, the supernatant was discarded and the residue was stained with 1% crystal violet solution containing 20% of ethanol. As to interferon potency, the dilution factor of the interferon sample showing the intensity of stain a half that of the difference in the intensity of stain between the virus-free well and the interferon-free, virus-added well was expressed in laboratory units/ml. This assay system affords a sensitivity 7 to 25 times as high as α-interferon for which an international standard is available. Whether the interferon activity was certainly human gamma-interferon activity was confirmed immunologically using an anti-human gamma-interferon antibody. In this case, 10 µl of anti-human gamma-interferon antibody (with a sufficient activity to inactivate 2,000 units/ml of human gamma-interferon activity) was added to 50 µl of an interferon active sample, and after standing at room temperature for 3 hours, the mixture was centrifuged at 10,000 r.p.m. for 5 minutes. The supernatant was separated for use as a sample. As shown in Table 1, the transformed E . coli WA802/pGIF₄ and WA802/pGIF₅ were confirmed to have produced a human gamma-interferon-like substance under the control of the lactose operon, while the non-transformant (WA802/pKO703) did not show this activity. The E . coli WA802/pGIF₄ disrupted/centrifuged super-natant which showed interferon activity in the above example was added to Ultrogel AcA 54 (0.7 cm dia. x 47 cm high) and its molecular weight was estimated by gel permeation chromatography, Physiologically balanced salt solution (1 x PBS) was used to obtain 0.7 ml fractions. As standard reference proteins, ribonuclease A (mol. wt. 13,700), chymotrypsinogen A (mol. wt. 25,000) and ovalbumin (mol. wt. 43,000) were employed. Interferon activity was eluted between chymotrypsinogen A and ovalbumin and the molecular weight of the peak interferon activity under the above conditions was estimated to be about 30,000 (See Fig. 12).

Claims: CLAIMS for the contracting states BE, CH, DE, FR, GB, IT, LI, LU, NL, SE A method of producing human gamma-interferon-like polypeptide which comprises cultivating in a nutrient medium host cells transformed with a plasmid with a polydeoxyribonucleotide, which has a translation-initiating codon at the 5'-end followed by a nucleotide sequence consisting of coding for the amino acid sequence and a subsequent nucleotide sequence coding for the termination of translation, inserted therein, and thereby causing formation of the polypeptide having the above amino acid sequence as a protein component in the culture broth. A method as claimed in claim 1 wherein the host cells are of a strain belonging to the genus Escherichia. A method as claimed in claim 2, wherein the host cells are of a strain belonging to the species Escherichia coli . A method as claimed in claim 3, wherein the host cells transformed are of Escherichia coli K12.WA802. A method as claimed in any preceding claim, wherein the gamma-interferon-like polypeptide is recovered from the culture broth. A polydeoxypoynucleotide of the sequence, A polydeoxyribonucleotide as claimed in claim 6, having in addition to said sequence a translation-initiating codon at the 5'-end and a translation-terminating codon at the 3'-end. A polydeoxyribonucleotide as claimed in claim 7, having restricted enzyme-cleavable nucleotide sequences added to both ends. A polydeoxyribonucleotide as claimed in claim 8 wherein the 5'-end side is cleavable with Eco RI and the 3'-end side with Sal I. A plasmid with a polydeoxyribonucleotide as defined in any one of claims 6 to 9 inserted therein. A strain belonging to Escherichia coli and transformed with a plasmid as claimed in claim 10. CLAIMS for the contracting state HT A method of producing human gamma-interferon-like polypeptide which comprises cultivating in a nutrient medium host cells transformed with a plasmid with a polydeoxyribonucleotide, which has a translation-initiating codon at the 5'-end followed by a nucleotide sequence consisting of and a subsequent nucleotide sequence coding for the termination of translation, inserted therein, and thereby causing formation of the polypeptide having the above amino acid sequence as a protein component in the culture broth. A method as claimed in Claim 1 wherein the host cells are of a strain belonging to the genus Escherichia. A method as claimed in Claim 2, wherein the host cells are of a strain belonging to the species Escherichia coli . A method as claimed in Claim 3, wherein the host cells transformed are of Escherichia coli K12.WA802. A method as claimed in any preceding claim, wherein the gamma-interferon-like polypeptide is recovered from the culture broth. A method as claimed in any preceeding claim wherein the said nucleotide sequence has restricted enzyme cleavable nucleotide sequences added to both ends. A method as claimed in Claim 6, wherein the 5'-end side is cleavable with Eco RI and the 3'-end side with Sal I.

Inventors: NAKASATO, HIROSHI, NOGUCHI, TERUHISA, OSHIMA, TAKEHIRO, TANAKA, SHOJI
Applicants: SUNTORY KABUSHIKI KAISHA
Application year: 1983
Main classification: C12N15
IPC codes: C12R1, C12N15, A61K38, C07H21, C07K14, C12P19, C12P21, C12N1