NOVEL HUMAN INTERFERON-GAMMA POLYPEPTIDE

Abstract: Disclosed is a novel human interferon-γ polypeptide, a recombinant plasmid wherein a DNA fragment coding for the polypeptide is incorporated, and a process for producing the human interferon-γ polypeptide using a microorganism con­ taining the plasmid.

Description: NOVEL HUMAN INTERFERON- yPOLYPEPTIDE Background of the Invention Interferons (referred to as IFN hereinafter) so far known can be classified into three large groups, i.e. TFN-c, IFN-ss and IFN-y. IFN-a is mainly produced by leukocytes, IFN-ss by fibroblasts and IFN-y by T-lymphocotes. These IFNs have been noted as biologically active substances having anti-virus activity, activating activitis upon natural killer cells and macrophages, anti-tumor activity, and the like However, the conventional methods for obtaining IFNs by isolation from leukocytes and cultured cells cannot provide them sufficiently, Recombinant DNA technology has now been developed to the extent that the mass production of substances which are secreted only in a small amount in cells of higher animals and are hard to isolate such as IFN has become possible by using microorganisms For example, mRNAG of IFN-ss and IFN-a were respectively isolated from cells and cDNA complementary to the mRNA was synthesized enzymatlcally, inserted into a suitable plasmid as a double stranded DNA and cloned in Escherichia coli [taniguchi, et al.: Proc. Jap. Acad., 55(B), 464-469 (1979), Nagata, et al. : Nature 294, 316-320 (1980)]. As for IFN-y, there has been a report that it has a stronger cell-inhibiting activity than other IFNs based on the experiment using animal cells [3.V Rubin and S L. Gupta: Proc. Natl. Acad. Sci., USA,-77, 5928-5932 (1980)). Furthermore, cloning of an IFN-y DNA into Escherichia coli and determination of it base sequence were recently reported rP.W. Gray, et al.: Mature 295, 503 (1982)]. The present inventors have made studies in order to provide a large amount of human IFN-Y polypeptide using recombinant DNA technology. As a result, it has been found that a novel IFN-y DNA with a base sequence different from that of the known IFN-y DNA incorporated in a recombinant plasmid is expressible in a microorganism, particularly in Escherichia coli Summary of the Invention The present invention relates to a novel human interleron-y polypeptide, a recombinant plasmid wherein a DNA fragment coding for the polypeptide is incorporated1 and a process for producing the human interferon-y polypeptide using a microorganism containing the plasmid. Brief Description of the Drawings Fig. 1 is a flow chart illustrating construction of plasmid pGC-7. Fig. 2 is a flow chart illustrating constrsction of plasmid pGKA-2. Fig. 3 illustrates the base sequence around the human IFN-y DNA in plasmid pGKA-2. Fig. 4 (A) illustrates the structure of plasmid pKYP-l and (B) illustrates that of pKYp-5 Fig. 5 is a flow chart illustrating cousiruction of pKYP-5. Fig. 6 is a flow chart illustrating construction of pRYP-lO. Fig. 7 (A) is a flow chart illustrating construc- tion of pKYP-ll and (B) illustrates the structure of pKYP- 12. Detailed Description of the Invention An object of the present invention is to provide a recombinant plasmid wherein a novel IFN-y DNA is incorporated, a microorganism containing the plasmid, a process for producing a novel human IFN-Y polypeptide using the microorganism and the human IFN-y polypeptide per se. Construction of the recombinant plasmid is carried out in the following manner. As the novel human IFN-y DNA, sIFNy-G4 which was clones by the present inventors is used. Escherichia coli containing pIFNr-G4 has been deposited with the American Type Culture Collection, U.S.A. under accession number ATCC 39123. The base sequence of the IFN-y DNA in pTENy-G4 was determined by the method of Maxam and Gilbert [Proc. Natl. Acad. Sci. 74, 560 (1977)] and is illustrated in Fig. 3. Comparison of the human IFN-ycDNA in pIFN&gamma;-G4 and the known IFE-ycDNA [P.W. Gray, et al.: Nature 295, 503 (1982)] reveals the following. The first base of the triplet coding for the ninth amino acid from the N-terminal of the mature human IFN-y polypeptide, lysine, in the known cDNA is adenine [Gray et al.: Nature 295, 503 (1982)]. On the other hand, as the corresponding base in the pIFN&gamma;- G4cDNA is cytosine, the ninth amino acid from the N-terminal of the human IFN-y polypeptide encoded by the pIFN&gamma;-G4cDNA is glutamine, and not sine. Therefore, it is apparent that pIFN&gamma;-G4 codes for a novel human IFN-y polypeptide. Further, sIrNy-G4 has a RsaI restriction site as the 25th base is C. That is, the sequence of the 22nd to 25th bases from 5'-terminus of the IiNy-DNA in pIFN&gamma;-G4 is GTAC and RsaI cuts the DNA at the position illustrated with the alrow $(Gr+AC). Since none of the known IFN-y genes have RsaI site in a DNA region coding for IFN-y polypeptide, it is apparent that pIFNy-Gd contains a DNA coding for a novel human IFN-y polypeptide. Therefore, recombinant plasmids constructed using IFN Y DNA of pIFN-&alpha;. G4 are all new recombinant plasmids. As the plasmid to incorporate IZ DLIA, any plasmid can be used so long as the DNA incorporated therein can be expressed in Escherichia coli. Preferably, 2 plasmid wherein a foreign DNA can be insert downstream from a suitable promoter such as trp promoter or lac promoter and the length of the sequenca between Shine-Dalgamo sequenco (reterred to as SD sequence hereinafter) and the initiation site of translation is adjusted to, for example, 6 - 18 base pairs is employed. Preferred examples are pKYP-10, pKYP-11 and pKYP-12 which were constructed by the present inventors [Japanese Patent Application 213193/81 (Japanese Published Ünexamined Patent Application 110600.83)]. These expression vectors prepared by the method illustrated in Reference Example 1 centain tryptophan promoters (referred to as Ptrp hereinafter). Recombinant plasmid pGC-7 is prepared frcm pKYP-11 and pIFN&gamma;-G4 as illustrated in Fig. 1. That is, pIFN&gamma;-G4 is digested with PuvII and HindIII and the digest is purified by low-gelling-temperature agarcse gel electrophoresis [E. wieslander, Analytical Biochemistry 98, 305 (1979]. The resulting fragment of pIFN&gamma;-G4 is inserted inte pKFP-11 at the HindIII- BemHI site to obtain pGC-7. Thereafter, as illustrated in Fig. 2, pGC-7 is cut with BstNI and SalI and IFN-&gamma; DNA is purified by Low-qellingterperature agarose gel electrophoresis. The IFN-&gamma; D is cloned together with a synthetic DNA in the HindIII - Sal site of the expression vector pKYP-10 harboring Ptrp to obtain recombinant plasmid pGKA-2. IFN-&gamma; polypcptide of the present invention is prepared in the following nanner. Escherichia coli K-12 HB101 [S.N. Cchen, ot al.: Proc. Natl. Acad. Sci., 69, 2110 (1972)]. is transformed with pGKA-2 and an Escharichia coli strain containing pGKA-2 is selected frcm colonies resistant to ampicillin (roderred to as ApR hereinarfter). The Escberichie coli serain con tainlng pGKA-2 is cultured in a medium to produce IT-y polypeptide in the cells. As the medium, either a synthetic medium or a natural medium can be used as long as it is suitable for the growth of Escherichia coli and the production of IFN-y polypeptide. As a carbon source, glucose, fructose, lactose, glycerol, mannitol, sorbitol, etc. may be used. As a nitrogen source, NH4Cl, (MH4)2SOd, casanino acid, yeast extract, polypeptone, meat extract, bactotryptone, corn steep liquor, etc. may be used. In addition, nutrients such as NH2HPO4, K2HPO4, KH2P04, NaCl, MgSO4, vitamine Bland MgCl2 may be used. Culturing is carried out at pH 5.5-B.5 and at 18 - 400C with aeration and stirring. After culturing for 5-90 hours, the human IFN-y polypeptide is accumulated in cultured cells. The collected cells are treated with lysozyme, disrupted by repeated freezing and melting and subjected to centrifugation. The thus obtained supernatant fluid is subjected to extraction according to a conventicnal method for extraction of polypeptides to recover the polypeptide. Determination of human IFN-&gamma; is carried out according to the method of Armstrong (J.A. Armstrong, et al.: Appl. Microbiol. 21, 723-725 (1971)]. Certain specific embodiments of the present invention are illustrated by the following examples. Example 1 Insertion of human IFN-y DNA into the expression vector pKYP-lI: In this example, 6 g of plasmid pIFNy-G4 [3.6 kilobases (rererred to as Kb hereinafter)] was dissolved in 50 p1 of a solution containing 20 mM Tris-ECl ( 7.5), 10 mM MgCl2, 10 rEL dithiothreitol and 50 mM NaCl. Then, 12 units each of restriction enzymes PvuII (product of Takara Shuzo Co.; the restriction enzymes hereinafter are all products of Takara Shuzo Co., unless otherwise specified) and HindIII were added and digestion reaction was carried out at 370C for 4 hours. The reaction solution was heated at 65 C for 7 minutes to inactivate the enzymes and subjected to purification by low-gelling-temperature agarose gel electrophoresis to obtain 1.2 ug of a DNA fragment containing human IFN-y DNA of 1.3 Kb. Separately, 4 g of pKYP-11 was dissolved in 40 l of a solution containing 20 mM Tris-HCl (pH 7.5) 10 mM MgCl2, 10 mM dithiothreitol and 50 mM NaCl, 8 units of BamHI was added and digestion reaction was carried out at 37 C for 3 hours. The reaction solution was heated at 650C for 5 minutes to inactivate the enzyme. Thereafter. 30 uM each of dATP, dCTP, dGTP and dTTP were added and 8 units of Escherichia coli DNA polymerase I (Klenow fragment, product of New England Biolabs, 1 l) was added. Fill-in reaction was carried out at 150C for 1 hour and the reac- tion solution was heated at 680C for 15 minutes to inacti vate the DNA polymerase 1. 10 units of HindIII was added and digestion reaction was carried out at 37 C for 3 hours, followed by heating at 65 C for 5 minutes to inactivate the HindIII. The digestion reaction solution of the plasmid pXYP-11 was subjected to purification by low-gelling-teoper ature agarose gel electrophoresis to obtain about 2,5 g o a DNA fragment of about 4.7 Kb containing Ptry. Then, 0.5 g of the DNA fragment of 1.3 Kb con t2lning human IFN-y DNA and 1.O g of the DNA fragment of about 4.7 Kb containing Ptrp, which was obtained from the plasmid pKYP-11, were dissolved in 20 pl of a solution contanning 20 taM Tris-HCl (pH 7.5), 6 mM MgCl2, 5 mM dibhio- threitol and 500 uM ATP, and 4 units of T4 DNA ligase (product of New England Biolabs) was added. Ligation reaction was carried out at 40C for 18 hours, and Escherich z coli HB101 was transformed with the obtained recombinant plasmid minture by conventional technigue to obtain an ApR colony. A plasmid, pGC-7 illustrated in Fig. 1 was separated rom the culture broth of the colony. The structure of pGC-7 was confirmed by digestion with HindIll, BamHI, HpaI, SalI, copi and Clal and agarose gel electrophoresis. Escherichia coli strain containing pGC-7 has been deposited with the Fermentation Research Institute, Agency of Industrial Science and Technology (referred to as FEP hereinafter) as Escherichia coli IGC-7 (FERM P-6814) (FERM BP-497). Example 2 Construction of recombinant plasmid pGKA-2: In this example, 6 g of the pGC-7 DNA obtained in Example 1 was dissolved in 50 l of a solution containing 20 mM Tris-GCl (pH 7.5). 10 mM MgCl2, 10 mM dithiothreitol and 10 mM NaCl, and 12 units of BstNI (product of New England Biolabs) was added. Reaction was carried out at 600C for 3 hours. Then, 150 mM NaCl and 8 units of SalI were added and digestion reaction was carried out at 370C fcr 3 hours. The reaction solution was again heated at 650C for 5 minutes to inactivate the salI and subjected to purification by low-gelling-temperature agarose gel electrophoresis to obtain about 0.8 g of a DNA fragment of about 1,125 base pairs (referred to as bp hereinafter) containing a large portion of the hun an IFN-y DNA. Separately, 3 ug of pKYP-10 was dissolved in 40 l of a solution containing 20 mM Tris-SCl (pH 7.50 10 mM MgCl2, 10 mM dithiothrcitol and 100 mM NaCl. 6 units cech of HindIII and Sell were added and digestion reaction was carried out at 370C for 3 hours. The redaction solution was heated at 65 C for 5 minutes to inactivate HindIII and SalI and subjected to purification by low-gelling-temperature agarose gel electrophoresis to obtain about 1.8 g of a DNA fragment of about 4.1 Rb containing Ptrp. The N-terminal amino acid of the mature human IFN-y polypeptide is cystein (Cys). In order to express mature IFN-Y DNA, it is necessary to furnish an initiation codon (ATG) just before the 5'-terminal codon TGT (Cys) and further to adjust the length of the sequence beteeen SD-seguence and ATG to a suitable length of 6-1B bp. Therefore, the following DNA linker was synthesized. EMI8.1 <tb> HindIII <SEP> M <SEP> e <SEP> t <SEP> C <SEP> y <SEP> s <SEP> T <SEP> y <SEP> r <SEP> C <SEP> y <SEP> s <SEP> BstNI <tb> 5' <SEP> A <SEP> T <SEP> A <SEP> C <SEP> A <SEP> C <SEP> A <SEP> A <SEP> T <SEP> G <SEP> A <SEP> C <SEP> G <SEP> G <SEP> T <SEP> 5' <SEP> (18-mer) <tb> <SEP> 3'A <SEP> G <SEP> C <SEP> TT <SEP> A <SEP> T <SEP> G <SEP> T <SEP> G <SEP> T <SEP> T <SEP> A <SEP> C <SEP> T <SEP> G <SEP> C <SEP> C <SEP> 3 <SEP> '(15-mer) <tb> Tv4o single chain DNAs of 18-mer and 15-mer were synthesized by a conventional tri-ester method [R. Crea, et al.: Proc. Natl. Acad. Sci., 75, 5765 (1978)]. Then, 2 g each of the 18-mer and 15-mer DNAs were dissolved in 20 l of a solution containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 5 mM dithiothreitol, 0.1 mM EDTA and 1 mM ATP. 30 units of T4 polynucleotide kinase (product of Boehringer Mannheim) was added and phosphorylation reaction was carried out at 370C for 60 minutes. Then, 2 g each or phosphorylated 18-mer and 15mer DNAs were mixed and the mixture was heated at 700C for 5 minutes and allowed to stand at room temperature for annealing to obtain the DNA linker having the structure given above 0.4 'Lg of the BstNI - Sali fragment of 1,125 b obtained above and derived from pGC-7 and 1.0 pg of the DNA ragment of 4.1 Kb obtained by digestion of the expression vector pKYP-10 with HindiIII and SalI were dissolves in 25 l of a solution containing 20 mM Tris-HCl (pH 7.5). 6 mM MgCl2, 5 mM dithiothreitol and 500 pM ATP. About 0.1 pg of the DNA linker mentioned above was added to the mixture, followed by addition of 6 units. of T4DNA ligase. Ligation reaction was carried out at 4 C for 17 hours. Escherichia coli HB101 was transformed using the obtained recombinant plasmid mixture by conventional technique to obtain an R colon. A plasmid; pGKA-2 illustrated in Fig. 2 was isolated from the culture broth of the colony. The struc ture of pGKA-2 was confirmed by digestion with EcoRI, Colad, HindIII, BstNI and SalI and agarose gel electrophoresis, it was confirmed by the method of Maxam-Gilbert [A.M. Maxam, et al.: Proc. Natl. Acad. Sci., 74, 560 (1977)] that the base sequence from the SD-sequence (AAG) to the initaiation codon (ATG) in the plasmid pGKA-2 is "AAGGGTA TCGATAAGCTTATG". The human IFN-y DNA in pGKA-2 is different from known DNAs in that the DNA has RsaI site and the ninth amino acid of the human IFN-y polypeptide encoded by the DMA is glutamine (Gln). Further, the synthesized DNA used above is different from the one used by P.W. Gray, et al. having the following structure: EMI9.1 <tb> <SEP> m <SEP> e <SEP> tc <SEP> ys <SEP> ty <SEP> rc <SEP> ys <SEP> <tb> A <SEP> AT <SEP> TC <SEP> AT <SEP> <SEP> GT <SEP> GT <SEP> TA <SEP> TT <SEP> GT <SEP> C <tb> <SEP> G <SEP> TA <SEP> CA <SEP> CA <SEP> AT <SEP> AA <SEP> CA <SEP> GT <tb> in the underlined parts. Thus, pGKA-2 has a restriction site for BstNI, CCAGG, in the DNA region coding for human IFN-y and this feature also distinguishes pGKA-2 from known plasmids. Furthermore, the length and structure between the SD sequence and ATG are important because of influence on the expression of proteins in Escherichia coli. The base sequence between the SD-sequence and ATG in pGKA-2 is apparently different from that in the known recombinant plasmid pIFN-y trp48 (P.W. Gray, et al.) Escherichia coli containing pGKA-2 has ben deposited with the FERM as Escherichia coli TGKA-2 (FERM P-6798) (FERM BP-496). Example 3 Production of interferon by Escherichia coli IGC-7 and IGKA-2: Escherichia coli HB101 strains containing the recombinant plasmids pGC-7 and pGKA-2 obtained in Examples 1 and 2 (IGC-7 and IGKA-2, respectively) were cultured at 37 C for 18 hours in an LG medium consisting of 10 g/l tryptone, 5 g/l yeast extract, 5 g/l NaCl and 2 g/l glucose and adjusted to pH 7.0 with NaOH, Then, 0.2 ml of the culture medium was inoculated in 10 ml of an MCG medium (pH 7.2) consisting of 0.6% Na2HPO4, 0.3% KH2PO4, 0.5% NaCI, 0.1% NH4C1, 0.5% glucose, 0.5% casamino acid, 1 mM MgSO4 and 4 g/ml vitamin B1. Culturing was carried out at 300C for 4-8 hours. Then, 10 g/ml indol acrylic acid (referred to as IAA hereinafter) which is an inducer of the tryptophan gene was added and culturing was continued for an additional 2-12 hours. Cells were harvested by centrifugation at 8,000 rpm for 10 minutes and washed with 30 mM NaCl and 30 mM Tris-HCl (pH 7.5) buffer solution. The washed cells were suspended in 1 ml of the buffer solution mentioned above and 200 tig of lysozyme and 5 p1 of 0.25 M EDTA (ethylene diamine tetraacetic acid) were added. The mixture was allowed to stand at 0 C for 30 minutes. Freezing and melting were repeated three times to disrupt the cells. The disrupted cells were centrifuged at 15,000 rpm for 30 minutes to obtain a supernatant fluid. The amount of the interferon in the supernatant was determined according to the mete of Armstrong [J.A. Armstrong, et al.: Appl. Microbiol. 21, 723 - 725 (1971)] wherein Sindvis virus was used as a virus and FL cell derived from human amnion cells was used as an animal cell. The result is shown in Table 1. Table 1 EMI10.1 <tb> Strain <SEP> Plasmid <SEP> contained <SEP> IFN-&gamma; <SEP> <SEP> (unit/l) <tb> IGC-7 <SEP> pGC-7 <SEP> 0 <tb> IGKA-2 <SEP> pGKA-2 <SEP> 1 <SEP> x <SEP> 106 <SEP> - <SEP> 2 <SEP> x <SEP> 108 <tb> Reference example 1 Construction of a plasmid vector having a trp promoter: (a) Purification of tryptophan transducing phage DNA: A #ptrp phage, #cIa57trpED10 [referred to as #trp. ED hereinafter, G.F. Miazzari, et al.: J. Bacteriol. 133, 1457 (1978)] was lysogenized in Escherichia coli JA 194 strin [F, #. Ik' mk' #trp E5, leu6, J. Carbon, et al.: Recombinant Molecules p.355 (1977), Raven Press]. The resultant lysogenic strain JA 194 (!trp ED) was cultured at 420C for 30 minutes.to induce #trp ED phaces and prepare phage lysate. The #phages were purified from the Aphage lysate by the cesium chloride equilibrium density gradient centrifugation or Yamakawa, et al.: "Chemicals of Nucleic Acids I" Tokyo Kagaku Dojin, p.54 - 61, (1974). The Aphages wer further purified bv phenol treatment and chloroform treatment according to the method of Yemakawa, et al.: "Chemicals of Nucleic Acids I" Tokyo Kagaku Dojin, p. 62 - 65, (1974) (b) Cloning of the trp promoter into a plasmid: Cloning of the trp operon from Atrp ED phage DNA was carried out in the following manner. That is, 8 g of Atrp ED DNA was digested at 370C for 2 hours with 16 units of EcoRI and 16 units of HindIII in 20 mI Tris-HCl (pH 7.5), 7S mM NaCl, 10 mM MgC12 and 5 mM dithiothreitol. Separately, 1 g of plasmid pbr325 DNA was digested with 2 units of EcoRI and 2 units of HindIII by the same method as mentioned above (final volume: 30 l). The reactions were stopped by heating at 65 C for 5 minutes. Then, 15 pL each of the digests were mixed and. 500 M (final concentration) ATP and 5 units of T4 DNA ligase (product of New England Biolabs) were added. The mixture was allowed to react at 40C for 18 hours. Escherichia coli C600 SF strain ±Cameron, et al.: Proc. Natl. Acad. Sci. 72, 3416 (1975)] was transformed with the plasmid mixture by the conventional method (S.N. Cohen, et al.: Proc. Natl. Acad. Sci. 5, 2110 (1972)] and transformants resistant to amplcillin (ApR), rersistant to tetracycline (Tcr) and sensitive to chloramphenicol (CmS) were obtained. Plasmid DNAs were isolated from the trans fo=nants of Escherichia coli and cleaved with EcoRI, HindIII and HpaI. One of the plasmid DNAs had the structure illustrated in Fig. 4 (A) and was named pK'LP-l. A TaqI cleavage site exists between 4 base pairs downstream from the SD sequence (AAGG) present downstream from the tryptophan promoter. By taking advantage of this fact, the plasmid pKYP-1 was digested with TagI and EcoRI and the digest was subjected to purification b agarose gel electrophoresis to obtain a 2.6 Kb DNA fragment containing the tryptophan promoter and SD sequence. The 2.6 Kb DNA fragment was cloned in a known vector, pBR322, by the method as illustrated in Fig. 5. That is, 8 g of pBR322 was digested at 45 C for 60 minutes with 2 units of Tal in 100 l of a reaction mixture comprising 10 mM Tris HC1 (pH 8.4), 6 mM MgCl2. 100 mM NaCl and 6 mM 2-mercaptoethanol. After partial digestion with TaqI, the digest was subjected to low-gelling-temperature agarose gel electrophoresis [Lars Wieslander: Analytical Biochemistry 98, 305 (1979)] to obtain a 4.36 Kb purified DNA fragment. About 1.5 g of the DNA fragment was completely digested at 37 C for 3 hours with 3 units of EcoRI, About 1.0 g of a 4.33 Kb DNA fragment was recovered by the same low gelling-temperature agarose gel electrophoresis as mentioned above. Then, 12 g of pKYP-1 DNA was partially digested with 3 units of TaqI, the digest was subjected- to low-gelling-temperature agarose gel electrophoresis to obtain about 2 us of a 8.5 Kb purified DNA fragment and the DNA was completely digested with EcoRI by the same procedure as above t obtain about 0.5 ug of a purified 2.6 kb DNA fragment. Then, O. g of the 4.33 Kb DNA of pBR322 and 0.25 g of the 2.6 Kb DNA fragment of pKYP-1 thus obtained were added to 20 l of a reaction solution consisting of 20 mM Tris HCl (pH 7.6), 10 mM MgCl2 and 10 mM dithiothreitol Then. 0.5 mM ATP and 4 units of T4 DNA ligase were added to the mixture and reaction was carried out at 4 C or 18 hours. Escherichia coli C600 SF8 strain was transformed with the recombinant plasmid DNA obtaincd as ahove. The pladmids in the transformant having ApR and Tcr were isolated. The plasmid DNA was digcstrd with 6 restriction endonucleases, EcoRI, Hindu, , ClaI (product of Boehringer Mannheim GmbH), Hpal, Hincil and BamHI to analyze the structure of the plasmid. The plasmid was recognized to have the structure illustrated in Fig. 4 (B) and was named pKYP-5. (c) Preparation of a portable promoter from the trp promoter: The plasmid vector pKYP-5 mentioned above is applicable as a DNA introducing vector since it has a ClaI site and a HindIII site on the DNA of 1 to 20 base pairs downstream from the SD sequence. However, another Clal site is present in pKYP-5 DNA besides the Clal site immediately after the SD sequence and the fragment with the trp promoter obtained by cutting pKYP-5 DNA with EcoRI and HindIII is a little too large, i.e. 2.65 Kb. For convenience of use, pKYP-5 was improved by the process as illustrated in Fig. 6 to obtain a plasmid having a DNA fragment containing a shorter tryptophan promoter. That is, pKYP-5 DNA was digested with HpaII and HindlIl and the digest was purified to obtain a DNA fragment of about 340 bp. The fragment was inserted into the pBR322 digested with ClaI and HindIII as illustrated in Fig. 6 to obtain pKIP-10. The structure of pKYP-10 was determined by digestion with EcoRI, Clay, HindIII and HpaI and agarose gel electrophoresis. (d) Connection of two or morn trp promoters: In order to construct a plasmid vector having a stronger prcmoter activity, two trp promoters were inserten into the vector. That is, the DNA fragment of about 340 bp containing the trp promoter mentioned in step (c) above was inserted into the pK'tP-10 digested with Clal and HindIII to obtain pKYP-l1 as illustrated in Fig. 7 (A). The same procedure was repeated to construct pKYP-12 illustrated in Fig. 7 (B) wherein three trp Promoters were connected at the same orientation. The structure of pKYP-12 was confirmed by digestion with rcoRI, Clal, Hindlil and Hpal.

Claims: WHAt IS CLAI1ED IS: 1. Human interferon-y polypeptide. 2. The polypeptide according to claim 1, wherein the polypeptide has the amino acid sequence illustrated in Fig. 3. 3. A recombinant plasmid wherein a DNA fragment coding for human interferon-y polypeptide is incorporated and its derivatives. 4. The recombinant plasmid according to claim 3, wherein the DNA fragment is incorporated downstream from a tryptophan promoter. 5. The recombinant plasmid according to claim 3 or 4, wherein the DNA fragment has the base sequence illus tratd in Fig. 3. 6. The recombinant plasmid according to claim 5 which is named pGC-7 or pGK.-2. 7. A process for producing human interferon-y polypeptide, which comprises culturing in a medium a udcreorganism transformed with a recombinant plasmid wherein a DNA fragment coding for human sinterferon-&gamma; polypeptide is incorporated, accumulating human interferon-y polypeptide in the culture medium and recovering human interferon-&gamma; polypeptide therefrom. 8. The process according to claim 7, wherein the DNA fragment is incorporated downstream from a tryptophan promoter. 9 The Process according to claim 7 or 8, wherein the microorganism belongs to Escherichia coli. 10. The process according to claim 7, 8 or 9, wherein the human interferon-&gamma; polypeptide has the amino acid sequence illustrated in Fig. 3. 11. A microorganism containing a recombinant plasmid wherein a DNA fragment coding for human interferon-y polypeptide is incorporated. 12. The microorganism according to claim 11, wherein the DNA fragment is incorporated downstream from a tryptophan promoter. 13. The microorganism according to claim 11 or 12 which belongs to Escherichia coli. 14. The microorganism according to claim 11, 12 or 13, wherein the DNA fragment has the base sequence illustrated in Fig. 3. 15. Escherichia coli iGC-7 (FERM P-6814) (FERM BP-437). 16. Escherichia coli IGKA-2 (FERM P-6798) (FEEMBP-496).

Inventors: ITOH, SEIGA, NISHI, TATSUNARI, SUGANO, HARUO, TANIGUCHI, TADATSUGU
Applicants: JURIDICAL FOUNDATION JAPANESE FOUNDATION FOR CANCER RESEARCH, KYOWA HAKKO KOGYO CO., LTD.
Application year: 1984
Main classification: C12N15
IPC codes: C12N15, A61K38, C12P21, C12N1, C07K14, C07H21, C12R1