NOVEL, PHYSIOLOGICALLY ACTIVE POLYPEPTIDES AND PRODUCTION METHOD THEREOF

Abstract: A novel polypeptide having the amino acid sequence: wherein X is a hydrogen atom or a methionyl group and Y is a peptide residue consisting of 2-10 amino acid residues of which at least the first two are basic amino acid residues. The polypeptide has high antiviral activity as compared with polipeptide consisting of protein portion of human gamma interferon, and can be produced by cultivating a transfor­ mant sigle cell microorganism obtained by transformation with an expression vector with the gene coding for the above-mentioned polypeptide inserted therein so as to the gene can be expressed.

Description: Novel, physiologically active polypeptides and production method thereof This invention relates to novel, physiologically active polypeptides and a method of producing the same. The physiologically active poEypeptides according to the invention carry the amino acid sequence contained in mature human gamma interferon (also called immune interferon) but are novel polypeptides differing therefrom in physico-chemical properties and having very high antiviral and antiproliferative activities as compared therewith. The invention also relates to a method of producing said novel polypeptides using recombinant DNA technology as well as cultures containing said polypeptides. Interferons are classified into three types, namely types a, ss and y, depending on their physicochemical properties and immunological properties, among others. All interferons are substances each consisting of a polypeptide and having an antiviral activity. Among them, y-type interferon (also called immune interferon) has potent cell growth inhibiting activity and therefore the use thereof as an antitumor or immunoregulating agent as well as the use as an antiviral agent has attracted much attention. As far as human gamma interferon (hereinafter abbreviated as "hIFN-y") is concerned, its amino acid sequence was shown at the Second Annual International Congress for Interferoii Research held at San Francisco, USA in October, 1981. Later, the DNA sequence of the human gene coding for said amino acid sequence was also revealed (R. Devos et al., Nucl. Acids Res., 10: 2487, 1982 and P. W. Gra et al., Nature, 295: 503, 1982). Furthermore, a patent application claiming a method of producing pre-hlFN-y and mature hIFN-y using a cDNW prepared based on a human lymphocyte-derived mRNA has been laid open (Japanese Kokai Tokkyo Koho No. 58-90514). Natural hIFN-y derived from human lymphocytes or spleen cells is a glycoprotein. Its physico-chemical characteristics, however, are not yet clear in many respects. The protein portion of mature hIFN-y has been shown, by using the recombinant DNA technology, to be a polypeptide composed of 146 amino acids. Nevertheless, the true structure of the active site or C-terminal site (e.g. whether the C-terminal amino acid is in the free form or in some or other modified form) is yet unknown. As a result of their intensive study of the effects of the active site and C-terminal site of hIFN-y consisting of 146 amino acids (hereinafter abbrevEated as "GIF146") on the physiological activities, the present inventors found that the polypeptide portion from the first to the 135th amino acid (hereinafter abbreviated as "GfF135"), in particular, is much superior in physiological activity to GIF)46 and differs also in physico-chemical properties tltrefrom and that said GIF135 polypeptide can be produced by recombinant DNA technology using a chemically syntllesized gene. TIie above surprising findings have now led to the present invention. The polypeptides of the present invention as represented by GIF135 can be extracted from cultivated transformant cells more easily as compared with G3F145 and moreover are characterized by higher stability. Therefore, they can be regarded as novel, physiologically active substances different from the prior art GIF146. Thus, the invention provides novel, physiologically active polypeptides represented by the amino acid sequence: 1 10 X Cys Tyr Cys Gln Asp Pro Tyr Val Lys Glu 20 Ala Glu Asn Leu Lys Lys Tyr Phe Asn Ala 30 Gly His Ser Asp Val Ala Asp Asn Gly Thr 40 Leu Phe Leu Gly Ile Leu Lys Asn Trp Lys 50 Glu Glu Ser Asp Arg Lys Ile Met Gln Ser 60 Glii lie Val Ser Phe Tyr Phe Lys Leu Phe 70 Lys Asn Phe Lys Asp Asp Gln Ser Ile Gln 80 Lys Ser Val Glu Thr lie Lys Glu Asp Met 90 Asn Val Lys Phe Phe Asn Ser Asn Lys Lys 100 Lys Arg Asp Asp Phe Glu Lys Leu Thr Asn 110 Tyr Ser Val Thr Asp Leu Asn Val Gln Arg 120 Lys Ala Ile His Glu Leu Ile Gln Val Met 130 Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly Y wherein X is a hydrogen atom or a methionyl group and Y is a peptide residue consisting of 2-10 amino acid residues of which at least the first two are basic amino acid residues, DNA sequences coding therefor, expression vectors in which any of said sequences is connected with a DN-A- sequence or sequences capable of causing efficient expression of the above polypeptide, single cell microorganism transformants obtained by transformation with said vectors, a method of producing the above polypeptides by cultivating said transfomants, and cultures of the above transformants which contain the above polypeptides. Taking Git135 as an example, the present invention is described below in detail. Referring to the drawings, Fig. 1 is a schematic representation of the process of constructing a GIF135 polypeptide expression vector, namely pYtacGIF135; Fig. 2 is a schematic representation of the process of constructing another GIF135 polypeptide expression vector, namely plNGIF135; Fig. 3 is a schematic representation of a method of modifying the base sequence corresponding to the neighborhood of the carboxyl terminal of GIF146 for the construction of a vector for GIF135 production. Among the GIF135-producing strains according to the invention, one (WA802/pYtacGIF135) was obtained by transforming a strain of Escherichia coli with a plasmid obtained by insertion of a chemically synthesized DNA fragment downstream from a chemically synthesized, GIF146-encoding gene inserted in the plasmid ptacGIF54Ci9 (disclosed in Japanese Patent Application No. 58-162337) and further insertion of a runnaway plasmid DNA (cf. Fig. 1). Another GIF-producing strain (WA802/pINGIF135 or W3110/pINGIF135) can be obtained by transforming the Escherichia coli strain WA802 or W3110 with a plasmid, pINGIF135, obtained by ligation of a BglII-PstI DNA fragment (i9 gene-containing fraction) from the above ptacGIF135 (Fig. 1) with a BglII-PstI DNA fragment (containing the ç promoter and the GIF gene) from pIN5GIF54C (disclosed in Fig. 4 of Japanese Patent Application No. 58-132524) (for said ligation, cf. Fig. 2). WA802/pYtacGIF135 is now described in more detail. First, the plasmid ptacGIF54Ciq was treated with the restriction enzymes BglII and SalI to thereby remove a DNA fragment (DNA fragment from the site coding for the 134th amino acid Arg to the SalI cleavage site just downstream from the translation termination codon) occurring downstream from the GIF-encoding gene. In place of said DNA fragment, a chemically synthesized DNA fragment having the BglII and SalI cohesive ends and two translation termination codons (TAA and TAG), namely GA TCT TAA TAG A ATT ATC AGC T was inserted, whereby the plasmid ptacGIF135 containing a GIF135-encoding gene was constructed (cf. Fig. 2). Then, the plasmid pYtacGIF135 was construct by introducing into the plasmid constructed above the runaway DNA from pYM307 and used for transformation of Escherichia coli WA802. The transformant thus obtained (WA802/pYtacGIF135), when compared with the transformant (WA802/pYtacGIF) with the GIF146 gene-containing plasmid pYtacGIF with respect to antiviral activity, showed a surprisingly high antiviral activity (cf. Table 1). pYtacGIF135 is a plasmid having the same DNA sequence as the plasmid pYtacGIF except for the downstream region of the hIFN-y gene. The host cell used is also the same. Nevertheless, the Escherichia coli strain WA802/pYtacGIF135 obtained by transformation wit the GIF135 gene-containing plasmid according to the invention, as the data given in Table 1 indicate, exhibited a surprisingly high antiviral activity as compared with Eschericia coli WA802/pYtacGIF, the transformant with pYtacGIF. Thus, cells of the transformant as obtained by growing it on L-broth containing ampicillin and kanamycin [induced culture using isopropyl B-Dthiogalactoside (IPTG)] were disrupted by freezing and thawing, followed by centrifugation. The supernatant (sup) thus obtained was about 35 times higher in antiviral activity and a fraction (ppt) obtained by extraction of the centrifugation residue with 8 M urea and 0.5 M NaCl about 96 times higher in said activity (about 73 times in total activity). Since the plasmids carried by both the transformants differ only in the hIFN-y gene portion and the productions of the polypeptides (GIF146 and Git135) are estimable to be theoretically equal, the results presented in Table 1 strongly suggest that GIF135 according to the invention is a polypeptide having very strong physiological activity as compared with the prior art GIF146. The antiviral activity ratio (sup/ppt) between the supernatant (sup) obtained by cell disruption and centrifugation and the fraction (ppt) obtained by extraction of the sediment with 8 M urea and 0.5 M NaCl is 0.62 for pYtacGIF whereas the same ratio is 0.23 for pYtacGIF135. It is therefore supposed that GIF135 and G1F146 also differ in physico-chemical properties (e.g. adsorbability to the cell membrane or cell wail (cf. Table 1). Furthermore, a distinct difference in solubility was observed. For instance, GIF135 is fairly soluble in urea solutions whereas only solutions containing urea plus a sot (e.g. sodium chloride) can dissolve G1F146 to a considerable extent. Table 1 Comparison in antiviral activity Transformant Antivial activity (IU/ml) sup/ppt sup*1 ppt*2 Total WA802/pYtacGIF 4,700 7,600 12,300 0.62 WA802/pYtacGIF135 166,600 728,200 894,700 0.23 (Notes] *1: Supernatant fraction obtained by centrifugation following disruption of cells by freezing and thawing. *2: Fraction obtained by extraction of the sediment obtained in the above centrifugation with 8 M urea and 0.5 M NaCl. The above facts indicate that the polypeptide according to the invention is not only superior in the physiological activity aspect but also provided with favorable physico-chemical properties distinct from those of Git146. In view of the structure of GIF146, it is evidently unobvious that the polypeptide of the invention has favorable properties such as mentioned above in spite of the fact that said polypeptide is included in the polypeptide structure of Git146. The polypeptide of the invention is thus very interesting as a novel antiviral, antitumor or immunoregulating agent. Analysis by electrophoresis and amino acid analysis have successfully demonstrated that the GIFl3S-producing strains according to the invention do produce the polypeptide consisting of 135 amino acid residues as expected. Thus, in amino acid analysis, the amino acid composition was in good agreement with the expected one and, furthermore, data suggesting that the carboxyl terminal portion of GIF135 is as expected could be obtained by carboxyl terminal analysis using carboxypeptidase. The following examples illustrate the invention are further detail. Although the following examples are illustrative of GIF135 and pYtacGIF135 alone, the scope of the present invention is by no means limited thereto. Examples 1. Production of GIF135 (1) Construction of ptacGIF135 In 50 us of 10 mM Tris-hydrochloric acid buffer (pH 7.4) containing 10 mM magnesium chloride, 150 mM sodium chloride and 10 mM 2-mercaptoethanol, there was dissolved 2 pg of the plasmid ptacGIF54Ci9 (disclosed in Fig. 3 of Japanese Patent Application No. 58-162337) DNA. To the solution were added 10 units of BglII and 1Q units of Sals. After incubation at 370C for 1 hour, the reaction mixture was subjected to agarose gel electrophoresis. The desired BglII-SalI fragment of about 7 kb was extracted electrophoporetically and treated with phenol, followed by addition of alcohol, whereby the desired Ca. 7 kb DNA fragment was obtained as a precipitate. Separately, 20 picomoles each of two ll-mer oligonucleotides (having the base sequences shown in Fig. 1 and Fig. 3, respectively) synthesized chemically by the solid phase method (cf. Japanese Patent Application No. 57-86180) were dissolved in 10 ul of 66 mM Tris-hydrochloric acid buffer (pH 7.6) containing 6.6 mM magnesium chloride. To the solution were added 1 p1 of 4 mM ATP and 2 units -of polynucleotide kinase, followed by incubation at 370C for 30 mintues for 5'-OH -phosphorylation. The approximately 7 kb BglII-SalI DNA fragment previously prepared was -dissolved in 10 p1 of 66 mM Tris buffer (pH 7.6) containing 6.6 mM magnesium chloride (hereinafter referred to as "ligation solution"), 10 p1 of the chemically synthesized DNA fragment solution was added thereto and, following addition of 1 unit of T4 DNA ligase, the ligation reaction was effected at 160C overnight. Using the resultant DNA, Escherichia coli WA802 was transformed in the conventional manner. The transformant selection was conducted using a nutrient agar medium containing 40 pg/ml of ampicillin and plasmid DNAs were isolated from 18 transformants out of a number ampicillin-resistant transformants obtained and analyzed using restriction enzymes. All the plasmids were found to be the contewmplated ptacGIF135. (2) Construction of pYtacGIF135 For the purpose of achieving increased expression of GIF135 through an increase in copy number, the longer EcoRI DNA fragment from the runaway plasmid pYM307 was inserted into ptacGIF135 at the EcoRI cleavage site thereof. The pYM307-carrying strain of Escherichia coli was named W620recA/pYM307. Said strain has been deposited with the Ferment ation Research Institute, Agency of Industrial Science and Technology under the deposit number FERM BP-345. First, 2 pg of ptacGIF135 DNA and 2 pg of pYM307 DNA were respectively dissolved in 50 p1 of 33 nss1 Tris-acetic acid buffer (pH 7.6) containing 66 mM potassium acetate, 10 mM magnesium acetate and 0.5 mM dithiothretiol, followed by addition of 5 units of EcoRI. After incubation at 370C for 1 hour, the restriction enzyme was inactivated by heating at 650C for 15 min & es and then ethanol was added. The thusobtained DNA precipitates were dried in vacuo and dissolved in 20 p1 of ligation solution and, following addition of 1 unit of T4 DNA ligase, the ligation was carried out at 160C overnight. The resultant DNA was used for transforming Escherichia coli WA802. The transformation was conducted at a temperature of 280C. Transformants were selected on a nutrient agar medium containing 40 pg/ml of ampicillin and 20 pg/ml of kanamycin. From among a number of ampicillin- and kanamycin-resistant transformants thus obtained, 12 strains were arbitrarily chosen and subjected to analysis using restriction enzymes and, as a result, all the strains analyzed could be confirmed to carry the intended plasmid pYtacGIF135. 2. Antiviral activity measurement For estimating the gamma interferon activity, WA802/pYtacGIF and WA802/pYtacGIF135 were respectively cultured in 2 ml of L-broth (containing 40 pg/ml of ampicillin) at 280C overnight and 0.2 ml of the cells obtained was inoculated into 4 ml of L-broth (containing 40 pg/ml of ampicillin). After incubation at 320C until the OD660 value reached about 0.7) 0.2 ml of 100 tnI IPTG solution was added and incubation was performed at 370C for 3 hours. A l-ml portion of the culture broth was centrifuged at 10,000 rpm for 5 minutes. To the bacterial cells thus obtained, there was added 500 1 of 0.15 M sodium phophate buffer (pH 7.2) containing 1 mg/ml lysozyme and 50 mM sodium chloride. The mixture was allowed to stand in an ice bath for 10 minutes. Following this bacteriolytic step, the cells were disrupted by three repetitions of rapid freezing in dry ice-ethanol and rapid thawing in a constant-temperature bath maintained at 370C. Centrifugation at 10,000 rpm for 10 minutes gave a supernatant, which was subjected to antiviral activity measurement. The antiviral activity was measured as disclosed in Japanese Patent Application No. 57-86180. For further recovering the desired polypeptide from the precipitate fraction, 500 p1 of 8 M urea solution contain;P3 0.15 mM sodium chloride was added to the precipitate, followed by sonication and centrifugation at 10,000 rpm for 10 minutes. The supernatant thus collected was found to have a very high antiviral activity. The results of such antiviral activity measurement are shown in Table 1. The fact that, as shown in Table 1, the Escherichia coli strain transformed with pYtacGIF135 produced a very high antiviral activity (about 73-fold higher in total activity) as compared with the same strain transformed with pTtacGIF in spite of the use of the same expression vector by the present inventors in the above example strongly suggests that GIF135 in accordance with the invention is a polypeptide having a surpassing antiviral activity which cannot be found in the so far known GIF146. 3. Amino acid analysis Polypeptide fractions were respectively extracted from the cultured cells of WA802/pYtacGIF135 and WA802/pYtacGIF by the method described in Example 2 and subjected to SDS gel electrophoresis. Gel fragments corresponding in molecular weight to GIF135 and GIF146 were excised, the respective polypeptides were extracted thereform, dialyzed against water to a sufficient extent and then subjected to amino acid analysis. The band corresponding to GIF135 on the SDS gel showed an evident shift to the lower molecular weight side from the band corresponding to Git146 and both the bands were found at their respective expected sites (estimated on the molecular weight basis). (1) Analysis of amino acid composition An amount equivalent to about 1 nanomole of each polypeptide extract was evaporated to dryness under reduced pressure, 100 p1 of 6 N HC1 containing 0.1% phenol was added, and hydrolysis was effected under reduced pressure at 1100C for 24, 48 or 72 hours. Separately, a sample after perfonsK acid oxidation was hydrolyzed with 6 N HC1 at 1100C for 24 hours. Each hydrolyzate was subjected to amino acid analysis which was performed in the conventional amnner using an amino acid analyzer (Hitachi model 835-50). The results obtained are shown in Table 2. The values for Met (methionine) given in Table 2 suggest that both the polypeptides are not high in the content of the translation startpoint amino acid methionine if said amino acid occurs attached to the amino terminal thereof. Table 2 Amino acid analysis of GIF135 and GIF146 F Amino GIF135 GIF146 acid Found Theoretical Found Theoretical CysSO3HA 1.7 2 2.3 2 Asp 19.8 20 19.9 20 n Thr 5.0 5 5.1 5 Ser 10.1B 10 10.7 11 Glu 16.0 16 17.7 18 Pro 2.2 2 2.4 2 Gly 4.3 4 5.2 5 Ala 7.2 7 8.3 8 Val 7.8C 8 7.7 8 Met 3.0 3 3.8 4 Ile 6.8C 7 6.5 7 LeuD 9.0 9 10.0 10 Tyr 4.5 5 4.8 5 Phe 8.9 9 9.6 10 Lys 19.5 20 20.0 20 His 2.0 2 1.8 2 Arg 5.0 5 7.7 8 E trip 1 1 Notes: A: Cys was claculated as CysSO3H. B: Calculated by extrapolation to time 0 (zero). C: Value after 72 hours off hydrolysis. D: Values for amino acids other than Leu were calculated with the value for Leu as the basis. E: trp cannot be determined because it is decomposed under the conditions of this analysis. F: For GIF146, values after 24 hours of hydrolysis are given. (2) Analysis of carboxyl terminal The above sample extracted from the SDS gel electrophoretic band was subjected to appropriate pretreatment (adjustment of pH, volume, etc.). Thereafter, a solution of carboxypeptidase A or a mixture of carboxypeptidase A and B in an appropriate concentration was added. After the subsequent 4 hours of reaction at 370C, 0.02 N HC1 was added, the pH and volume were further adjusted with 70% formic acid and then amino acid analysis was performed by the conventional method. For the GIF135 sample treated with carboxypeptidase A and B, the amino acids detected were Ser (1.0), Arg (1.8), Lys (2.7), Gly (0.9), Thr (1.1) and Ala (1.0) alone (the numerical values given in the parentheses being based on the mole ratio values for the respective amino acids as obtained upon acid hydrolysis of the same GIF135 sample). For the sample treated with carboxypeptidase A, Ser (0.7), Arg (0.2) and Lys (0.1) were detected. The above results strongly suggest that the carboxyl terminal portion of GIF135 has the expected amino acid sequence. Japanese Patent Applications Nos. 57-86180, 58-132524 and 58-162337 referred to herein correspond to European Patent Applications Nos. 83302915.0, 84304901.6 and 84305996.5 respectively. Strains WA802 and w3110 are available from E. Coli Genetic Stock Control (USA).

Claims: Claims: 1. A physiologically active polypeptide having the amino acid sequence: X Cys Tyr Cys Gln Asp Pro Tyr Val Lys 10 Glu Ala Glu Asn Leu Lys Lys Tyr Phe 20 Asn Ala Gly His Ser Asp Val Ala Asp 30 Asn Gly Thr Leu Phe Leu Gly Ile Leu 40 Lys Asn Trp Lys Glu Glu Ser Asp Arg 50 Lys lie Met Gln Ser Gln Ile Val Ser 60 Phe Tyr Phe Lys Leu Phe Lys Asn Phe 70 Lys Asp Asp Gln Ser Ile Gln Lys Ser 0 Val Glu Thr Ile Lys Glu Asp Met Asn 90 Val Lys Phe Phe Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr 100 Asn Tyr Ser Val Thr Asp Leu Asn Val 110 Gln Arg Lys Ala Ile His Glu Leu Ile 120 Gln Val Met Ala Glu Leu Ser Pro Ala 130 Ala Lys Thr Gly Y wherein X is a hydrogen atom or a methionyl group and Y is a peptide residue consisting of 2-10 amino acid residues of which at least the first two are basic amino acid residues. 2. Ç physiologically active polypeptide of Claim 1, which has the amino acid sequence: X Cys Tyr Cys Gln Asp Pro Tyr Val Lys 10 Glu Ala Glu Asn Leu Lys Lys Tyr Phe 20 Asn Ale Gly His Ser Asp Val Ale Asp 30 Asn Gly Thr Leu Phe Leu Gly lie Leu 40 Lys Ann Txp Lys Glu Giu Ser Asp Arg 50 Lys Ile Met Gln Ser Gln lie Val Ser 60 Phe Tyr Phe Lys Leu Phe Lye Asn Phe 70 Lye Asp Asp Gln Ser Ile Gln Lys Ser 80 Val Glu Thr Ile Lye Glu Asp Met Asn 90 Val Lye Phe Phe Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr 100 Asn Tyr Ser Val Thr Asp Leu Asn Val 110 Gln Arg Lys Ala Ile His Glu Leu Ile 120 Gln Val Met Ala Glu Leu Ser Pro Ala 130 135 Ala Lys Thr Gly Lys Arg Lys Arg Ser wherein X is a hydrogen atom or a methionyl group. 3. A DNA sequence which codes for the polypeptide of Claim 1 or 2. 4. A DNA sequence of Claim 3, which is a product of chemical synthesis. 5. tr DNA sequence of Claim 3 or 4, which contains the DNA sequence: EMI19.1 AAG GAA GCT GAA AAC CTG AAG AAA TAC TTC CTT CGA CTT TTG GAC TTC TTT ATG TTC AAC GCT GGT CAT TCT GAC GTT GCT AAG TTG CGA CCA GTA AGA CTG CAA CGA GAC AAC GGT ACT CTG TTC CTG GGT ATC CTG TTG CCA TGA GAC AAG GAC CCA TAG CTG Mh MC TGG AM GM GM TCT GAC GAC TTT TTG ACC TTT CTT CTT AGA CTG CGT AAA ATC ATG CAG TCT CAG ATC GTT GCA TTT TAG TAC GTC AGA GTC TAG CM TCT TTC TAC TTC MG CTG TTC AAA AAC AGA AAG ATG AAG TTC GAC MG TTT TTG TTC AAG GAC GAC CAG TCT ATC CAG AAA AAG TTC CTG CTG GTC AGA TAG GTC TTT TCT GTT GAA ACT ATC AAG GAA GAC ATG AGA CAA CTT TGA TAG TTC CTT CTG TAC AAC GTT AAG TTC TTC AAC TCT AAC AAG TTG CAA TTC AAG AAG TTG AGA TTG TTC <RTI ID=20.4> AtA AAG CGT GAC GAC TTC GM AAG CTT TTT TTC GCA CTG CTG AAG CTT TTC GAA ACT AAC TAC TCT GTT ACT GAC CTT MT TGA TTG ATG AGA CAA TGA CTG GAA TTA GTA CAG CGT AAA GCT ATC CAT GAA CTG CAT GTC GCA TTT CGA TAC GTA CTT GAC ATC CAG GTT ATG GCT GAA CTG TCC CCG TAG GTC CM TAC CGA CTT GAC AGG GGC GCT GCT AAA ACT GGT AAG CGT MA AGA CGA CGA TTT TGA CCA TTC GCA TTT TCT TCT AGA wherein the sequence in the square EMI20.1 may alternatively TGT be ACA 6. A DNA sequence as claimed in any of Claims 3-5, wherein the DNA sequence comprises a DNA fragment containing an EcoRI cleavage site as connected upstream from said DNA sequence and a DNA fragment containing one or two termination codons (TAA or TAG) and a SalI cleavage site as connected downstream from said DNA sequence. 7. An expression vector in which the DNA sequence coding for the polypeptide of Claim 1 is effectively connected with a DNA sequence capable of causing expression of said polypeptide. 8. s' expression vector of Claim 7 which contains the tac promoter, which is a hybrid promoter between the Escherichia coli trp and lac promoters, the lactose repressor gene lac19 and a plasmid DNA having the function of increasing the plasmid copy number. 9. %n expression vector of Claim 7 which has a modification of the untranslated 5'-end region of the Escherichia coli lipoprotein-regulating structural gene. 10. An expression vector of Claim 8 which is selected from the group consisting of pYtacGIFl35 and ptacGIFl35 species. 11. n expression vector of Claim 8 which is selected from the group consisting of pINGIFl35 species. 12. A single cell microorganism transformant obtained by transformation with an expression vector with the gene coding for the polypeptide of Claim 1 inserted therein a manner such that said gene can be expressed. 13. A transformant of Claim 12, wherein said expression vector is the expression vector of any of Claims 7 to 11. 14. A transformant of Claim 13, wherein the microorganism transformed is a strain of Escherichia coli. 15. A transformant of Claim 14 which is selected from the group consisting of WAB02/pYtacGIFl35 and WA802/ptacGIFl35 strains. 16. tr transformant of Claim 14 which is selected from the group consisting of -6RA802/pINGIF135 and W3110/pINGIF135 strains. 17. A method of producing Cz polypeptide of Claim 1, which comprises cultivating the transformant of any of Claims 9 to 12 and harvesting said polypeptide. 18. A culture of Cx transformant of any of Claims 12 to 16, which contains the polypeptide of Claim 1. 19. A polypeptide as claimed in Claim 1 or Claim 2, for use as an antiviral, antitumor or immunoregulating agent in the treatment of the human or animal body. 20. An antiviral, antitumor or immunoregulating composition comprising a polypeptide as claimed in Claim 1 or Claim 2 as an antiviral, antitumor or immunoregulating agent.

Inventors: NOMURA, MIDORI, TANAKA, SHOJI
Applicants: SUNTORY KABUSHIKI KAISHA, ALSO KNOWN AS SUNTORY LTD.
Application year: 1984
Main classification: C07K13
IPC codes: C12N1, C07K14, A61K38, C12N15, A61P31, C12P21, C07H21, C12R1