Novel human interferon-gamma polypeptide derivative

Disclosed is a novel derivative of human interferon-.gamma. polypeptide, a recombinant plasmid wherein a DNA fragment coding for said polypeptide is incorporated and a process for producing the derivative of human interferon-.gamma. polypeptide using a microorganism containing said plasmid.

BACKGROUND OF THE INVENTION 
Interferons (referred to as IFN hereinafter) so far known can be classified 
into three large groups, i.e. IFN-.alpha., IFN-.beta. and IFN-.gamma.. 
IFN-.alpha. is mainly produced from leukocytes, IFN-.beta. from 
fibroblasts and IFN-.gamma. from T-lymphocytes. These IFNs have been noted 
as biologically active substances having anti-virus activity, activating 
activities 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, mRNAs of IFN-.beta. and 
IFN-.alpha. were respectively isolated from cells and DNA complementary to 
the mRNA (cDNA) was synthesized enzymatically and cloned in Escherichia 
coli [Taniguchi, et al.: Proc. Jap. Acad., 55 (B), 464-469 (1979), Nagata, 
et al.: Nature 284, 316-320 (1980)]. 
As for IFN-.gamma., there has been a report that it has a stronger 
cell-inhibiting activity than other IFNs based on the experiment using 
animal cells [B. Y. Rubin and S. L. Gupta: Proc. Natl. Acad. Sci., USA, 
77, 5928-5932 (1980)]. Furthermore, cloning of an IFN-.gamma. cDNA into 
Escherichia coli and determination of its base sequence were recently 
reported [P. W. Gray, et al.: Nature 295, 503 (1982), R. Devos, et al.: 
Nucleic Acids Research 10, 2487 (1982)]. 
The present inventors have independently cloned a DNA coding for 
IFN-.gamma. to obtain a clone coding for a novel IFN-.gamma. wherein, as 
apparent from the base sequence illustrated in Table 1, the ninth amino 
acid of the mature IFN-.gamma. reported by Devos, et al., Lysine (Lys) 
(AAA), is replaced with glutamine (Gln) (CAA). Further, the IFN-.gamma. 
gene was incorporated into vector pKYP-10 having tryptophan prompter 
(Japanese Published Unexamined Patent Application No. (110600/83) and mass 
production of the IFN-.gamma. in Escherichia coli has been achieved. 
Thereafter, the present inventors have studied the production of 
derivatives of IFN-.gamma. polypeptide using the IFN-.gamma. gene 
illustrated in Table 1 as a starting material. 
It was reported that deletion of 11 amino acids from the C-terminal of 
IFN-.alpha. decreased specific activity to one-third [A. E. Franke, et 
al.: DNA 1, 223-230 (1982)], whereas addition of 18 amino acids to the 
N-terminal of IFN-.alpha. did not change specific activity [R. M. King, et 
al.: J. Gen. Virol. 64, 1815-1818 (1983)]. 
Derivatives of IFN-.gamma. have not yet been reported. The present 
inventors have constructed a derivative wherein the third amino acid of 
IFN-.gamma. illustrated in Table 1, cysteine (Cys) was replaced with 
tyrosine (Tyr) (referred to as 3-Tyr-IFN-.gamma. hereinafter) and found 
that the specific activity was 2-4 times stronger than that of the parent 
IFN-.gamma.. Further, the derivatives wherein the Cys at the position 1 
was replaced with serine (Ser) (1-Ser-IFN-.gamma.), the Cys at the 
position 3 was replaced with Ser (3-Ser-IFN-.gamma.), the Cys at the 
positions 1 and 3 were replaced with Ser (1,3-Ser-IFN-.gamma.) and 
N-terminal amino acids of IFN-.gamma. illustrated in Table 1 were deleted 
were constructed. Equivalent or more interferon activity was detected for 
all the derivatives compared with the starting IFN-.gamma.. 
SUMMARY OF THE INVENTION 
The present invention relates to a novel derivative of human 
interferon-.gamma. polypeptide, a recombinant plasmid wherein a DNA 
fragment coding for said polypeptide is incorporated and a process for 
producing the derivative of human interferon-.gamma. polypeptide using a 
microorganism containing said plasmid. 
TABLE 1 
__________________________________________________________________________ 
CACATTGTTCTGATCAT CTGAAGATCAGCTATTAGAAG AGAAAGATCAGTTAAGTCCTTTGG 
ACCTGATCAGCTTGATACAAGA ACTACTGATTTCAACTTCTTTGG CTTAATTCTCTCGGAAACG 
##STR1## 
##STR2## 
##STR3## 
##STR4## 
##STR5## 
##STR6## 
##STR7## 
##STR8## 
##STR9## 
##STR10## 
CCTGCAATATTTGAATTTTAAATC TAAATCTATTTATTAATATTTAACAT TATTTATATGGGGAATATATTT 
TTAG 
ACTCATCAATCAAATAAGTATTTA TAATAGCAACTTTTGTGTAATGAAAA TGAATATCTATTAATATATGTA 
TTA 
TTTATAATTCCTATATCCTGT GACTGTCTCACTTAATCCTTTGTTTTCTGA CTAATTAGGCAAGGCTATGTG 
ATT 
ACAAGGCTTTATCTCAGGGGCCAACT AGGCAGCCAACCTAAGCAAGATCCCATG 
GGTTGTGTGTTTATTTCACTT 
##STR11## 
__________________________________________________________________________

DETAILED DESCRIPTION OF THE INVENTION 
An object of the present invention is to provide a recombinant plasmid 
wherein a DNA coding for a novel derivative of human IFN-.gamma. is 
incorporated, a microorganism containing the plasmid, a process for 
producing a novel derivative of human IFN-.gamma. polypeptide using the 
microorganism and the derivative of human IFN-.gamma. polypeptide per se. 
Construction of the recombinant plasmid is carried out using cDNA obtained 
from messenger RNA coding for IFN-.gamma. by recombinant DNA technology or 
chromosomal DNA coding for IFN-.gamma. as a starting material. 
In the present invention, any human IFN-.gamma. cDNA are employable and 
pIFN-.gamma.-G4 is preferably used. Escherichia coli containing 
pIFN.gamma.-G4 has been deposited with the American Type Culture 
Collection, USA under accession number ATCC 39123. 
The base sequence of the IFN-.gamma. DNA in pIFN.gamma.-G4 was determined 
by the method of Maxam and Gilbert [Proc. Natl. Acad. Sci. 74, 560 (1977)] 
and is illustrated in Table 1. 
Comparison of the human IFN-.gamma. cDNA in pIFN.gamma.-G4 and the known 
IFN-.gamma. cDNA [R. Devos, et al.: Nucleic Acids Research, 10, 2487 
(1982)] reveals the following. The first base [adenine (A)] of the triplet 
coding for lysine (the ninth amino acid of the mature human IFN-.gamma. 
polypeptide) in the known IFN-.gamma. is replaced by cytosine (C) in the 
pIFN.gamma.-G4 cDNA. Accordingly, the ninth amino acid from the N-terminal 
of the human IFN-.gamma. polypeptide encoded by the pIFN.gamma.-G4 cDNA is 
glutamine, and not lysine. Therefore, it is apparent that pIFN.gamma.-G4 
codes for a novel human IFN-.gamma. polypeptide. 
Derivatives of IFN-65 obtained by deletion or replacement of amino acids 
of IFN-.gamma. illustrated in Table 1 are also novel IFN-.gamma. 
derivatives. 
As the plasmid to incorporate a DNA coding for IFN-.gamma. derivative, any 
plasmid can be used so long as the DNA incorporated therein can be 
expressed in Escherichia coli. Preferably, a plasmid wherein a foreign DNA 
can be inserted downstream from a suitable promoter such as trp promoter 
or lac promoter and the length between Shine-Dalgarno sequence (referred 
to as SD sequence hereinafter) and initiation codon (ATG) is adjusted, for 
example, to 6-18 base pairs is employed. Preferred examples are pKYP10, 
pKYP11 and pKYP12 which were constructed by the present inventors 
(Japanese Published Unexamined Patent Application No. 110600/83). 
As illustrated in FIG. 1, pIFN.gamma.-G4 is cleaved with PvuII and BamHI 
linker is introduced into the cleavage site to obtain pGBD-1. 
Then, pGBD-1 is digested with SinI and BamHI and a fragment of about 850 bp 
is purified by low-gelling-temperature agarose gel electrophoresis (LGT 
method) [L. Wieslander: Analytical Biochemistry 98, 305 (1979)]. pKYP-10 
is digested with ClaI and BamHI and a fragment of about 4.3 kb is 
purified. The thus obtained DNA fragments and a synthetic DNA illustrated 
in FIG. 2 which codes for Tyr as the third amino acid are ligated with T4 
DNA ligase to obtain pGSB-6. Then, pGSB-6 is digested with ClaI and 
subjected to fill-in reaction with DNA polymerase I and ligation reaction 
with T4 DNA ligase to obtain pGVA-4. The same procedure is repeated except 
for using the synthetic DNA illustrated in FIG. 3 which codes for Ser as 
the first and third amino acids to obtain plasmid pGVK-13 which codes for 
a derivative wherein the N-terminal first and third amino acids of 
IFN-.gamma., Cys, are replaced with Ser. 
In order to obtain IFN-.gamma. derivative wherein N-terminal amino acids 
are deleted, as illustrated in FIG. 4, pGKA-2 is digested with ClaI, 
treated with Bal31 for a short period of 1-30 minutes to cut out the DNA 
coding for N-terminal amino acid of IFN-.gamma. and digested with PstI and 
a fragment of 4.3 Kb is purified. Separately, vector pTrS-3 containing 
initiation codon is digested with SphI, treated with DNA polymerase I and 
digested with PstI and a fragment of 880 bp is purified. Both fragments 
are ligated with T4 ligase to obtain pGWC-10. 
Reaction conditions required for the recombinant DNA technology described 
above are generally as follows. 
Digestion of the DNA with restriction enzymes is usually carried out by 
reacting 0.1 to 20 .mu.g of DNA with 0.1-100 units, preferably 1-3 units 
of restriction enzyme per 1 .mu.g of DNA in a mixture of 2-200 mM, 
preferably 10-40 mM Tris-HCl (pH 6.0-9.5, preferably pH 7.0-8.0), 0-200 mM 
NaCl and 2-20 mM, preferably 5-10 mM MgCl.sub.2 at 20.degree.-70.degree. 
C. (optimal temperature depends on restriction enzymes used) for 15 
minutes to 24 hours. Reaction is usually stopped by heating at 
55.degree.-75.degree. C. for 5-30 minutes, or alternatively by 
inactivating the restriction enzyme with the reagent such as phenol or 
diethylpyrocarbonate. 
Purification of the DNA fragments formed by digestion with restriction 
enzymes is carried out by LGT method or polyacrylamide gel electrophoresis 
[A. M. Maxam, et al.: Proc. Natl. Acad. Sci., USA 74, 560 (1977)]. 
Ligation of the DNA fragments is carried out with 0.3-10 units of T4 DNA 
ligase in a mixture of 2-200 mM, preferably 10-40 mM Tris-HCl (pH 6.1-9.5, 
preferably 7.0-8.0), 2-20 mM, preferably 5-10 mM MgCl.sub.2, 0.1-10 mM, 
preferably 0.5-2.0 mM ATP and 1-50 mM, preferably 5-10 mM dithiothreitol 
at 1.degree.-37.degree. C., preferably 3.degree.-20.degree. C. for 15 
minutes to 72 hours, preferably 2-20 hours. The recombinant plasmid DNA 
formed by the ligation reaction is introduced into Escherichia coli by the 
transformation method of Cohen, et al. [S. N. Cohen, et al.: Proc. Natl, 
Acad. Sci. USA 69, 2110 (1972)], if necessary. Isolation of the 
recombinant plasmid DNA from Escherichia coli carrying the DNA is carried 
out by the method described in Example 1 or the method of Birnboim, et al. 
[H. C. Birnboim, et al.: Nucleic Acids Res. 7, 1513 (1979)]. Plasmid DNA 
is digested with 1-10 kinds of restriction endonucleases and the cleavage 
sites are examined by agarose gel electrophoresis or polyacrylamide gel 
electrophersis. Further, if necessary, the base sequence of the DNA is 
determined by the method of Maxam-Gilbert [Proc. Natl. Acad. Sci. 74, 560 
(1977)]. 
The derivative of IFN-.gamma. polypeptide of the present invention is 
produced by the following method. 
That is, Escherichia coli K-12 HB101 is transformed with a plasmid such as 
pGVA-4 and an Escherichia coli strain carrying pGVA-4 is selected from the 
ampicillin resistant (referred to as Ap.sup.R hereinafter) colonies. The 
Escherichia coli strain carrying pGVA-4 is cultured in a medium to produce 
a derivative of IFN-.gamma. polypeptide in the cultured cells. 
As the medium, either a synthetic medium or a natural medium can be used so 
long as it is suitable for the growth of Escherichia coli and the 
production of the derivative of IFN-.gamma.polypeptide. 
As a carbon source, glucose, fructose, lactose, glycerol, mannitol, 
sorbitol, etc. may be used. 
As a nitrogen source, NH.sub.4 Cl, (NH.sub.4).sub.2 SO.sub.4, casamino 
acid, yeast extract, polypeptone, meat extract, Bactotrypton, corn steep 
liquor, etc. may be used. 
In addition, nutrients such as K.sub.2 HPO.sub.4, KH.sub.2 PO.sub.4, NaCl, 
MgSO.sub.4, vitamine B.sub.1 and MgCl.sub.2 may be used. 
Culturing is carried out at pH 5.5-8.5 and at 18.degree.-40.degree. C. with 
aeration and stirring. 
After culturing for 5-90 hours, the derivative of human IFN-.gamma. 
polypeptide is accumulated in cultured cells. The collected cells are 
treated with lysozyme, disrupted by repeated freezing and thawing and 
subjected to centrifugation. The thus obtained supernatant fluid is 
subjected to extraction according to a conventional 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 
Construction of plasmid pGBD-1 having BamHI cleavage site downstream from 
IFN-.gamma. gene: 
In this example, 2 .mu.g of plasmid pIFN.gamma.-G4 [3.6 kilobases (referred 
to as Kb hereinafter)] was dissolved in 50 .mu.l (total volume) of a 
solution containing 20 mM Tris-HCl (pH 7.5), 10 mM MgCl.sub.2, 10 mM 
dithiothreitol and 50 mM NaCl (referred as "Y-50 buffer solution" 
hereinafter). Then, 4 units of restriction enzyme PvuII (product of Takara 
Shuzo Co.; the restriction enzymes hereinafter are all products of Takara 
Shuzo Co., unless otherwise specified) was added and digestion reaction 
was carried out at 37.degree. C. for 2 hours. 1 .mu.g of DNA fragment (3.6 
Kb) of pIFN.gamma.-G4 was purified by LGT method. 0.1 .mu.g of the DNA 
fragment and 5 pmoles of 5'-phosphorylated BamHI linker 
(5'-pCCGGATCCGG-3': product of Collaborative Research, Inc.) were ligated 
at 4.degree. C. for 18 hours with 2 units of T4 ligase (product of Takara 
Shuzo Co.; the same shall apply hereinafter) in 20 .mu.l of a buffer 
solution consisting of 20 mM Tris-HCl (pH 7.6), 10 mM MgCl.sub.2, 10 mm 
dithiothreitol and 0.5 mM ATP (referred to as "T4 ligase buffer solution" 
hereinafter). 
Escherichia coli HB101 [Boliver, et al.: GENE 2, 75 (1977)] was transformed 
using the thus obtained recombinant plasmid DNA by the method of Cohen, et 
al. [S. N. Cohen, et al.: Proc. Natl. Acad. Sci. USA. 69, 2110 (1972), the 
method is used for transformation of Escherichia coli hereinafter] to 
obtain an Ap.sup.R colony. Plasmid DNA was isolated from the transformant 
by the known method [H. C. Birnboim, et al.: Nucleic Acids Res., 7, 1513 
(1979), this method is used for isolation of plasmid DNA hereinafter]. The 
DNA was digested with restriction endonucleases such as BamHI and its 
structure was analyzed to recognize that recombinant plasmid pGBD-1 
wherein BamHI linker was inserted into PvuII site of pIFN.gamma.-G4 was 
obtained. Escherichia coli strain carrying plasmid pGBD-1 has been 
deposited with the Fermentation Research Institute, Agency of Industrial 
Science and Technology (referred to as FERM hereinafter) as Escherichia 
coli IGBD-1 (FERM BP-394). 
EXAMPLE 2 
Construction of recombinant plasmid pGVA-4 coding for 3-Tyr-IFN-.gamma.: 
pGBD-1 obtained in Example 1 was treated by the following method to remove 
the modification of DNA and make SinI cleavage site in the plasmid. 
Escherichia coli GM31 (thr leu dcm his thi ara lac galK, galT, xyl mtl str 
tonA) [Marinus, et al.: Molec. Gen. Genet. 127, 47-55 (1973)] was 
transformed with pGBD-1 by a conventional method. A large amount of pGBD-1 
was prepared from the thus obtained colonies by a conventional method. 6 
.mu.g of the thus obtained pGBD-1 DNA was dissolved in 50 .mu.l of Y-50 
buffer solution. 10 units of SinI (product of Bio Tech Co.) was added and 
digestion reaction was carried out at 37.degree. C. for 3 hours. Then, 
NaCl was added to a final concentration of 100 mM and 10 units of BamHI 
was added. Digestion reaction was carried out at 37.degree. C. for 3 
hours. About 0.8 .mu.g of DNA fragment of about 850 base pairs (referred 
to as bp hereinafter) containing the most part of human IFN-.gamma. DNA 
was obtained from the reaction solution by LGT method. 
Separately, 3 .mu.g of pKYP10 prepared by the method described in Japanese 
Published Unexamined Patent Application No. 110600/83 was dissolved in 40 
.mu.l (total volume) of a buffer solution consisting of 20 mM Tris-HCl (pH 
7.5), 10 mM MgCl.sub.2, 10 mM dithiothreitol and 100 mM NaCl (referred to 
as "Y-100 buffer solution" hereinafter) and 5 units each of ClaI (product 
of Boehringer Mannheim GmbH) and BamHI were added. Digestion reaction was 
carried out at 37.degree. C. for 3 hours. From the reaction solution, 
about 1.8 .mu.g of DNA fragment of about 4.3 Kb containing tryptophan 
promoter (P.sub.trp) was obtained by LGT method. 
Mature human IFN-.gamma. polypeptide has the N-terminal structure of 
Cys-Tyr-Cys-. In order to change the third amino acid (Cys) to Tyr and to 
furnish an initiation codon (ATG) necessary for expression just before the 
first Cys, the following DNA linker was synthesized. 
##STR12## 
Two single chain DNAs of 17-mer and 18-mer were synthesized by a 
conventional triester method [R. Crea. et al: Proc. Natl. Acad. Sci., 75, 
5765 (1978)]. Then, 2 .mu.g each of the 17-mer and 18-mer DNAs were 
dissolved in 40 .mu.l (total volume) of a solution containing 50 mm 
Tris-HCl (pH 7.5), 10 mM MgCl.sub.2, 5 mM dithiothreitol, 0.1 mM EDTA and 
1 mM ATP. 30 units of T4 polynucleotide kinase (product of Takara Shuzo 
Co.) was added and phosphorylation reaction was carried out at 37.degree. 
C. for 60 minutes. 
0.4 .mu.g of the SinI-BamHI fragment of about 850 bp obtained above and 
derived from pGBD-1 and 1.0 .mu.g of the ClaI-BamHI fragment of about 4.3 
Kb of the expression vector pKYP10 obtained above were dissolved in 25 
.mu.l of T4 ligase buffer solution. About 0.1 .mu.g of the DNA linker 
mentioned above was added to the mixture, followed by addition of 6 units 
of T4 DNA ligase. Ligation reaction was carried out at 4.degree. C. for 17 
hours. 
Escherichia coli HB101 was transformed using the obtained recombinant 
plasmid mixture to obtain an Ap.sup.R colony. A plasmid pGSB-6 illustrated 
in FIG. 2 was isolated from the culture broth of the colony. The structure 
of pGSB-6 was confirmed by the digestion with EcoRI, ClaI, and BamHI and 
agarose gel electrophoresis. It was confirmed by the method of 
Maxam-Gilbert [A. M. Maxam, et al.: Proc. Natl. Acad. Sci., USA 74, 560 
(1977)] that the base sequence around ClaI-SinI in the plasmid pGSB-6 is 
##STR13## 
The human IFN-.gamma. polypeptide encoded by pGSB-6 (the derivative is 
named 3-Tyr-IFN-.gamma. hereinafter) is clearly different from the known 
human IFN-.gamma. polypeptide in that the third amino acid (Cys) of mature 
human IFN-.gamma. is replaced with Tyr. 
Then, 1 .mu.g of the pGSB-6 was dissolved in 30 .mu.l (total volume) of 
Y-50 buffer solution and 2 units of ClaI was added. Digestion reaction was 
carried out at 37.degree. C. for 2 hours. A DNA fragment was recovered by 
phenol extraction, chloroform extraction and ethanol precipitation. The 
DNA fragment was dissolved in 30 .mu.l of a solution consisting of 67 mM 
Tris-HCl (pH 8.8), 6.7 mM MgCl.sub.2, 10 mM mercaptoethanol, 6.7 .mu.M 
EDTA, 16.6 mM (NH.sub.4).sub.2 SO.sub.4, 1 mM dATP, 1 mM dCTP, 1 mM dGTP 
and 1 mM dTTP and 5 units of T4 DNA polymerase (product of Takara Shuzo 
Co.) was added, followed by fill-in reaction at 37.degree. C. for 1 hour. 
A DNA fragment was recovered by phenol extraction, chloroform extraction 
and ethanol precipitation. 0.1 .mu.g of the DNA fragment was dissolved in 
50 .mu.l of T4 ligase buffer solution. 2 units of T4 ligase was added and 
ligation reaction was carried out at 4.degree. C. for 16 hours. 
Escherichia coli HB101 was transformed using the thus obtained DNA mixture 
and plasmid DNA, pGVA-4, was recovered from the formed Ap.sup.R colony. 
The structure of pGVA-4 was recognized by the cleavage with NruI, BamHI 
and EcoRI. The base sequence between SD and ATG of pGVA-4 was confirmed by 
the method of Maxam and Gilbert described above and is illustrated below. 
##STR14## 
Escherichia coli strain carrying plasmid pGVA-4 has been deposited with the 
FERM as Escherichia coli IGVA-4 (FERM BP-395). 
EXAMPLE 3 
Construction of recombinant plasmid pGVK-13 coding for 1,3-Ser-IFN-.gamma.: 
6 .mu.g of pGBD-1 DNA obtained in Example 1 was dissolved in 50 .mu.l of 
Y-50 buffer solution. 10 units of SinI (product of Bio Tec Co.) was added 
and digestion reaction was carried out at 37.degree. C. for 3 hours. Then, 
NaCl was added to a final concentration of 100 mM and 10 units of BamHI 
was added. Digestion reaction was carried out at 37.degree. C. for 3 
hours. About 0.8 .mu.g of DNA fragment of about 850 bp containing the most 
part of human IFN-.gamma. DNA was obtained from the reaction solution by 
LGT method. Separately, 3 .mu.g of pKYP-10 was dissolved in 40 .mu.l 
(total volume) of Y-100 buffer solution and 5 units each of HindIII and 
BamHI were added. Digestion reaction was carried out at 37.degree. C. for 
3 hours. From the reaction solution, about 1.8 .mu.g of DNA fragment of 
about 4.3 Kb containing P.sub.trp was obtained by LGT method. 
Mature human IFN-.gamma. polypeptide has the N-terminal structure of 
Cys-Tyr-Cys-. In order to change the first and third amino acids (Cys) to 
Ser and to furnish an initiation codon (ATG) necessary for expression just 
before the first Ser, the following DNA linker was synthesized. 
##STR15## 
Two single chain DNAs of 20-mer and 19-mer were synthesized by a 
conventional triester method [R. Crea, et al.: Proc. Natl. Acad. Sci., 75, 
5765 (1978)]. Then, 2 .mu.g each of the 20-mer and 19-mer DNAs were 
dissolved in 40 .mu.l (total volume) of a solution containing 50 mM 
Tris-HCl (pH 7.5), 10 mM MgCl.sub.2, 5 mM dithiothreitol, 0.1 mM EDTA and 
1 mM ATP. 30 units of T4 polynucleotide kinase (product of Takara Shuzo 
Co.) was added and phosphorylation reaction was carried out at 37.degree. 
C. for 60 minutes. 
0.4 .mu.g of the SinI-BamHI fragment of about 850 bp obtained above and 
derived from pGBD-1 and 1.0 .mu.g of the HindIII-BamHI fragment of about 
4.3 Kb of the expression vector pKYP10 obtained above were dissolved in 25 
.mu.l of T4 ligase buffer solution. About 0.1 .mu.g of the DNA linker 
mentioned above was added to the mixture, followed by addition of 6 units 
of T4 DNA ligase. Ligation reaction was carried out at 4.degree. C. for 17 
hours. 
Escherichia coli HB101 was transformed using the obtained recombinant 
plasmid mixture to obtain an Ap.sup.R colony. A plasmid, pGVK-13 
illustrated in FIG. 3 was isolated from the culture broth of the colony. 
The structure of pGVK-13 was confirmed by the digestion with EcoRI, 
HindIII, ClaI, and BamHI and agarose gel electrophoresis. It was confirmed 
by the method of Maxam-Gilbert that the base sequence from HindIII site to 
SinI site in the plasmid pGVK-13 is as follows. 
##STR16## 
The human IFN-.gamma. polypeptide encoded by pGVK-13 (the derivative is 
named 1,3-Ser-IFN-.gamma. hereinafter) is clearly different from the known 
human IFN-.gamma. polypeptide in that the first and third amino acids 
(Cys) of mature human IFN-.gamma. are replaced with Ser. Escherichia coli 
strain carrying plasmid pGVK-13 has been deposited with the FERM as 
Escherichia coli IGVK-13 (FERM BP-432). 
EXAMPLE 4 
Construction of plasmid pGWC-10 coding for the polypeptide wherein an 
N-terminal region of human IFN-.gamma. is deleted: 
25 .mu.g of pGKA-2 (5.2 Kb) obtained by the method of Reference Example 2 
was dissolved in 400 .mu.l of a buffer solution consisting of 20 mM 
Tris-HCl (pH 7.5), 10 mM MgCl.sub.2, 10 mM dithiothreitol and 10 mM NaCl 
(referred to as "Y-10 buffer solution" hereinafter). 50 units of ClaI 
(product of Boehringer Mannheim GmbH) was added and digestion reaction was 
carried out at 37.degree. C. for 3 hours. To 80 .mu.l of the reaction 
solution containing 5 .mu.g of DNA, were added 12 .mu.l of 10-fold 
concentrated BAL31 buffer solution [200 mM Tris-HCl (pH 8.1), 1M NaCl, 120 
mM CaCl.sub.2 ], 28 .mu.l of water and 0.25 unit of nuclease BAL31 
[product of Bethesda Research Laboratories (BRL)] and reaction was carried 
out at 30.degree. C. for 20 seconds. BAL31 has the activity of exonuclease 
which digests from the end of a DNA molecule and about 30 base pairs of 
DNA from ClaI site were digested under the conditions described above. DNA 
was recovered from the reaction solution by phenol extraction, chloroform 
extraction and ethanol precipitation. 1.0 .mu.g of the thus recovered 
pGKA-2 fragment digested with ClaI and BAL31 was dissolved in 20 .mu.l of 
Y-50 buffer solution. 2 units of PstI was added and digestion reaction was 
carried out at 37.degree. C. for 2 hours. From the reaction solution, 0.5 
.mu.g of DNA fragment of about 4.3 Kb was recovered by LGT method. 
Then, 5.0 .mu.g of ATG expression vector pTrS3 (3.8 Kb) was dissolved in 40 
.mu.l of Y-50 buffer solution. 10 units of SphI (product of BRL) was added 
and digestion reaction was carried out at 37.degree. C. for 3 hours. After 
phenol extraction and chloroform extraction, about 3.0 .mu.g of DNA 
fragment was recovered by ethanol precipitation. About 3.0 .mu.g of the 
DNA fragment was dissolved in a solution consisting of 67 mM Tris-HCl (pH 
8.3), 6.7 mM MgCl.sub.2, 10 mM mercaptoethanol, 6.7 .mu.M EDTA and 16.6 mM 
(NH.sub.4).sub.2 SO.sub.4 and 1 mM each of dATP, dTTP, dCTP and dGTP were 
added. Further, 6 units of T4 DNA polymerase (product of Takara Shuzo Co., 
the same shall apply hereinafter) was added and reaction was carried out 
at 37.degree. C. for 1 hour to digest the protruding end. After phenol 
extraction and chloroform extraction, 1.0 .mu.g of DNA fragment was 
recovered by ethanol precipitation. 1.0 .mu.g of the DNA fragment was 
dissolved in 20 .mu.l (total volume) of Y-50 buffer solution. 2 units of 
PstI was added and digestion reaction was carried out at 37.degree. C. for 
3 hours. From the reaction solution, 0.5 .mu.g of DNA fragment of about 
880 bp containing P.sub.trp was recovered by LGT method. 
0.5 .mu.g of ClaI-PstI fragment (about 4.3 Kb) of pGKA2 obtained above and 
0.5 .mu.g of PTrS3-SphI-T4 polymerase-PstI fragment (880 bp) obtained 
above were dissolved in 10 .mu.l (total volume) of T4 ligase buffer 
solution. 0.3 unit of T4 DNA ligase was added and ligation reaction was 
carried out at 4.degree. C. for 18 hours. 
Escherichia coli HB101 was transformed using the thus obtained recombinant 
plasmid mixture and plasmid DNA was recovered from the Ap.sup.R colony 
formed to obtain pGWC-10 illustrated in Fig. 5. The structure of pGWC-10 
was recognized by the digestion with EcoRI, ClaI and BamHI and agarose gel 
electrophoresis. It was confirmed by the method of Maxam and Gilbert that 
the sequence around N-terminal of human IFN-.gamma. structure gene in 
pGWC-10 was as follows. 
##STR17## 
It was also confirmed that seven amino acids from the N-terminal amino 
acid (Cys) to the seventh amino acid (Tyr) of the mature human IFN-.gamma. 
polypeptide were deleted and that the human IFN-.gamma. polypeptide 
derivative was started with the eighth amino acid (Val) [the derivative is 
named IFN-.gamma. (.DELTA.1-7)]. Escherichia coli strain carrying plasmid 
pGWC-10 has been deposited with the FERM as Escherichia coli IGWC-10 (FERM 
BP-397). 
EXAMPLE 5 
Production of IFN-.gamma. derivatives by Escherichia coli strains carrying 
pGBD-1, pGVA-4, pGVK-13 and pGWC-10: 
Escherichia coli HB101 strains carrying recombinant plasmids pGBD-1, 
pGVA-4, pGVK-13 and pGWC-10 obtained in Examples 1-4, which are named 
IGBD-1, IGVA-4, IGVK-13 and IGWC-10 respectively, were cultured at 
37.degree. C. for 18 hours in LG medium (10 g of trypton, 5 g of yeast 
extract, 5 g of NaCl, 2 g of glucose, 1 l of water, adjusted to pH 7.0 
with NaOH). 0.2 ml of the culture broth was inoculated into 10 ml of MCG 
medium (0.6% Na.sub.2 HPO.sub.4, 0.3% KH.sub.2 PO.sub.4, 0.5% NaCl, 0.1% 
NH.sub.4 Cl, 0.5% glucose, 0.5% casamino acid, 1 mM MgSO.sub.4, 4 .mu.g/ml 
vitamine B.sub.1, pH 7.2) and culturing was carried out at 30.degree. C 
for 4 hours. Then, 10 .mu.g/ml indolacrylic acid (referred to as IAA 
hereinafter) which is an inducer of tryptophan operon was added and 
culturing was continued for 4 hours. The culture broth was centrifuged at 
8,000 rpm for 10 minutes and the cells harvested were washed with a buffer 
solution containing 30 mM NaCl and 30 mM Tris-HCl (pH 7.5). Washed cells 
were suspended in 1 ml of the buffer solution described above and 5 .mu.l 
of a solution containing 200 .mu.g of lysozyme and 0.25M EDTA 
(ethylenediamine tetraacetic acid) was added. The mixture was allowed to 
stand at 0.degree. C. for 30 minutes and freezing and thawing 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 interferon in the supernatant was determined according to 
the mothod of Armstrong [J. A. Armstrong, et al.: Appl. Microbiol. 21, 
723-725 (1971)], wherein Sindvis virus was used as the virus and FL cells 
derived from human amnion cells were used as the animal cells. The results 
are shown in Table 2. 
TABLE 2 
______________________________________ 
Product encoded 
IFN-.gamma. 
Strains Plasmid by the plasmid 
(units/ml) 
______________________________________ 
IGBD-1 pGBD-1 IFN-.gamma. trace 
IGVA-4 pGVA-4 3-Tyr--IFN-.gamma. 
9 .times. 10.sup.4 
IGVK-13 pGVK-13 1,3-Ser--IFN-.gamma. 
2 .times. 10.sup.5 
IGWC-10 pGWC-10 IFN-.gamma.(.DELTA.1-7) 
5 .times. 10.sup.4 
IGKA-2 pGKA-2 IFN-.gamma. 2 .times. 10.sup.4 
______________________________________ 
IGKA-2 is a strain carrying plasmid pGKA-2 coding for IFN-.gamma.. 
EXAMPLE 6 
Construction of recombinant plasmid pGVL10 coding for 1-Ser-IFN-.gamma.: 
6 .mu.g of pGBD1 DNA obtained in Example 1 was dissolved in 50 .mu.l of 
Y-50 buffer solution. 10 units of SinI was added and digestion reaction 
was carried out at 37.degree. C. for 3 hours. Then, NaCl was added to a 
final concentration of 100 mM and 10 units of BamHI was added. Digestion 
reaction was carried out at 37.degree. C. for 3 hours. About 0.8 .mu.g of 
DNA fragment of about 850 bp containing the most part of human IFN-.gamma. 
DNA was obtained from the reaction solution by LGT method. 
Separately, 3 .mu.g of pKYP10 DNA prepared by the method described in 
Japanese Published Unexamined Patent Application No. 110600/83 was 
dissolved in 40 .mu.l (total volume) of Y-50 buffer solution and 5 units 
each of HindIII and BamHI were added. Digestion reaction was carried out 
at 37.degree. C. for 3 hours. From the reaction solution, about 1.8 .mu.g 
of DNA fragment of about 4.3 Kb containing tryptophan promoter (P.sub.trp) 
was obtained by LGT method. 
Mature human IFN-.gamma. polypeptide has the N-terminal structure of 
Cys-Tyr-Cys-. In order to change the first Cys to Ser and to furnish an 
initiation codon (ATG) necessary for expression just before the first Ser, 
the following DNA linker was synthesized. 
##STR18## 
Two single chain DNAs of 20-mer and 19-mer were synthesized by a 
conventional triester method [R. Crea, et al.: Proc. Natl. Acad. Sci., 
USA, 75, 5765 (1978)]. Then, 2 .mu.g each of the 20-mer and 19-mer DNAs 
were dissolved in 40 .mu.l (total volume) of a solution containing 50 mM 
Tris-HCl (pH 7.5), 10 mM MgCl.sub.2, 5 mM dithiothreitol, 0.1 mM EDTA and 
1 mM ATP. 30 units of T4 polynucleotide kinase was added and 
phosphorylation reaction was carried out at 37.degree. C. for 60 minutes. 
0.5 .mu.g of the SinI-BamHI fragment of about 850 bp obtained above and 
derived from pGBD-1 and 1.0 .mu.g of the HindIII-BamHI fragment of about 
4.3 Kb of the expression vector pKYP-10 were dissolved in 25 .mu.l of T4 
ligase buffer solution. About 0.1 .mu.g of the DNA linker mentioned above 
was added to the mixture, followed by addition of 6 units of T4 DNA 
ligase. Ligation reaction was carried out at 4.degree. C. for 17 hours. 
Escherichia coli HB101 was transformed using the obtained recombinant 
plasmid mixture to obtain an Ap.sup.R colony. A plasmid, pGVL10 
illustrated in FIG. 5 was isolated from the culture broth of the colony. 
The structure of pGVL10 was confirmed by the digestion with EcoRI, ClaI, 
HindIII and BamHI and agarose gel elecrophoresis. It was confirmed by the 
method of Maxam-Gilbert [A. M. Maxam, et al.: Proc, Natl, Acad, Sci. USA, 
74, 560 (1977)] that the base sequence from HindIII site to SinI site in 
the plasmid pGVL10 is as follows. 
##STR19## 
The human IFN-.gamma. polypeptide encoded by pGVL10 (the derivative is 
named 1-Ser-IFN-.gamma. hereinafter) is clearly different from the known 
human IFN-.gamma. polypeptide in that the first Cys of mature human 
IFN-.gamma. is replaced with Ser. Escherichia coli strain carrying plasmid 
pGVL10 has been deposited with the FERM as Escherichia coli IGVL10 (FERM 
BP-544). 
EXAMPLE 7 
Construction of recombinant plasmid pGVM101 coding for 3-Ser-IFN-.gamma.: 
6 .mu.g of pGBD1 DNA obtained in Example 1 was dissolved in 50 .mu.l pf 
Y-50 buffer solution. 10 units of Sin I was added and digestion reaction 
was carried out at 37.degree. C. for 3 hours. Then, NaCl was added to a 
final concentration of 100 mM and 10 units of BamHI was added. Digestion 
reaction was carried out at 37.degree. C. for 3 hours. About 0.8 .mu.g of 
DNA fragment of about 850 bp containing the most part of human IFN-.gamma. 
DNA was obtained from the reaction solution by LGT method. 
Separately, 3 .mu.g of pKYP10 DNA prepaed by the method described in 
Japanese Published Unexamined Patent Application No. 110600/83 was 
dissolved in 40 .mu.l (total volume) of Y-50 buffer solution and 5 units 
each of HindIII and BamHI were added. Digestion reaction was carried out 
at 37.degree. C. for 3 hours. From the reaction solution, about 1.8 .mu.g 
of DNA fragment of about 4.3 Kb containing tryptophan promoter (P.sub.trp) 
was obtained by LGT method. 
Mature human IFN-.gamma. polypeptide has the N-terminal structure of 
Cys-Tyr-Cys-. In order to change the third amino acid (Cys) to Ser and to 
furnish an initiation codon (ATG) necessary for expression just before the 
first Cys, the following DNA linker was synthesized. 
##STR20## 
Two single chain DNAs of 20-mer and 19-mer were synthesized by a 
conventional triester method. Then, 2 .mu.g each of the 20-mer and 19-mer 
DNAs were dissolved in 40 .mu.l (total volume) of a solution containing 50 
mM Tris-HCl (pH 7.5), 10 mM MgCl.sub.2, 5 mM dithiothreitol, 0.1 mM EDTA 
and 1 mM ATP. 30 units of T4 polynucleotide kinase was added and 
phosphorylation reaction was carried out at 37.degree. C. for 60 minutes. 
0.5 .mu.g of the SinI-BamHI fragment of about 850 bp obtained above and 
derived from pGBD1 and 1.0 .mu.g of the HindIII-BamHI fragment of about 
4.3 Kb of the expression vector pKYP10 were dissolved in 25 .mu.l of T4 
ligase buffer solution. About 0.1 .mu.g of the DNA linker mentioned above 
was added to the mixture, followed by addition of 6 units of T4 DNA 
ligase. Ligation reaction was carried out at 4.degree. C. for 17 hours. 
Escherichia coli HB101 was transformed using the obtained recombinant 
plasmid mixture to obtain an Ap.sup.R colony. A plasmid, pGVM101 
illustrated in FIG. 6 was isolated from the culture broth of the colony. 
The structure of pGVM101 was confirmed by the digestion with EcoRI, ClaI, 
HindIII, and BamHI and agarose gel electrophoresis. It was confirmed by 
the method of Maxam-Gilbert that the base sequence from HindIII site to 
SinI site in the plasmid pGVM101 is as follows. 
##STR21## 
The human IFN-.gamma. polypeptide encoded by pGVM101 (the derivative is 
named 3-Ser-IFN-.gamma. hereinafter) is clearly different from the known 
human IFN-.gamma. polypeptide in that the third amino acid (Cys) of mature 
human IFN-.gamma. is replaced with Ser. Escherichia coli strain carrying 
plasmid pGVM101 has been deposited with the FERM as Escherichia coli 
IGVM101 (FERM BP-545). 
EXAMPLE 8 
Construction of plasmid pGWE4 coding for the polypeptide wherein an 
N-terminal region of human IFN-.gamma. is deleted: 
25 .mu.g of pGKA2 (5.2 Kb) obtained by the method of Reference Example 2 
was dissolved in 400 .mu.l of Y-10 buffer solution. 50 units of ClaI was 
added and digestion reaction was carried out at 37.degree. C. for 3 hours. 
To 80 .mu.l of the reaction solution containing 5 .mu.g of DNA, were added 
12 .mu.l of 10-fold concentrated BAL31 buffer solution, 28 .mu.l of water 
and 0.25 unit of nuclease BAL31 and reaction was carried out at 30.degree. 
C. for 10 seconds. BAL31 has the activity of exonuclease which digests 
from the end of a DNA molecule and about 20 base pairs of DNA from ClaI 
side were digested under the conditions described above. DNA was recovered 
from the reaction solution by phenol extraction, chloroform extraction and 
ethanol precipitation. 1.0 .mu.g of the thus recovered pGKA2 fragment 
digested with ClaI and BAL 31 was dissolved in 20 .mu.l of Y-50 buffer 
solution. 2 units of PstI was added and digestion reaction was carried out 
at 37.degree. C. for 2 hours. From the reaction solution, 0.5 .mu.g of DNA 
fragment of about 4.3 Kb was recovered by LGT method. 
Then, 5.0 .mu.g of ATG expression vector pTrS3 (3.8 Kb) was dissolved in 40 
.mu.l of Y-50 buffer solution. 10 units of SphI was added and digestion 
reaction was carried out at 37.degree. C. for 3 hours. After phenol 
extraction and chloroform extraction, about 3.0 .mu.g of DNA fragment was 
recovered by ethanol precipitation. About 3.0 .mu.g of the DNA fragment 
was dissolved in a solution consisting of 67 mM Tris-HCl (pH 8.3), 6.7 mM 
MgCl.sub.2, 10 mM mercaptoethanol, 6.7 .mu.M EDTA and 16.6 mM 
(NH.sub.4).sub.2 SO.sub.4 and 1 mM each of dATP, dTTP, dCTP and dGTP were 
added. Further, 6 units of T4 DNA polymerase was added and reaction was 
carried out at 37.degree. C. for 1 hour to digest the protruding end. 
After phenol extraction and chloroform extraction, 1.0 .mu.g of DNA 
fragment was recovered by ethanol precipitation. 1.0 .mu.g of the DNA 
fragment was dissolved in 20 .mu.l (total volume) of Y-50 buffer solution. 
2 units of PstI was added and digestion reaction was carried out at 
37.degree. C. for 3 hours. From the reaction solution, 0.5 .mu.g of DNA 
fragment of about 880 bp containing P.sub.trp was recovered by LGT method. 
0.5 .mu.g of ClaI-PstI fragment (about 4.3 Kb) of pGKA2 obtained above and 
0.5 .mu.g of pTrS3-SphI-T4 polymerase-PstI fragment (880 bp) obtained 
above were dissolved in 10 .mu.l (total volume) of T4 ligase buffer 
solution. 0.3 unit of T4 DNA ligase was added and ligation reaction was 
carried out at 4.degree. C. for 18 hours. 
Escherichia coli HB101 was transformed using the thus obtained recombinant 
plasmid mixture and plasmid DNA was recovered from the Ap.sup.R colony 
formed to obtain pGWE4 illustrated in FIG. 7. The structure of pGWE4 was 
recognized by the digestion with EcoRI, ClaI and BamHI and agarose gel 
electrophoresis. It was confirmed by the method of Maxam and Gilbert that 
sequence around N-terminal of human IFN-.gamma. structure gene in pGWE4 
was as follows. 
##STR22## 
It was also confirmed that four amino acids from the N-terminal amino acid 
(Cys) to the fourth amino acid (Gln) of the mature human IFN-.gamma. 
polypeptide were deleted and that the human IFN-.gamma. polypeptide 
derivative was started with the fifth amino acid (Asp) [the derivative is 
named IFN-.gamma.(.DELTA.1-4)]. Escherichia coli strain carrying plasmid 
pGWE4 has been deposited with the FERM as Escherichia coli IGWE4 (FERM 
BP-546). 
EXAMPLE 9 
Production of IFN-.gamma. derivatives by Escherichia coli strains carrying 
pGVL10, pGVM101 and pGWE4: 
Escherichia coli HB101 strains carrying recombinant plasmids pGVL10, 
pGVM101 and pGWE4 obtained in Examples 6-8, which are named IGVL10, 
IGVM101, and IGWE4 respectively, were cultured at 37.degree. C. for 18 
hours in LG medium. 0.2 ml of the culture broth was inoculated into 10 ml 
of MCG medium and culturing was carried out at 30.degree. C. for 4 hours. 
Then 10 .mu.g/ml IAA was added and culturing was continued for 4 hours. 
The culture broth was centrifuged at 8,000 rpm for 10 minutes and the 
cells harvested were washed with a buffer solution containing 30 mM NaCl 
and 30 mM Tris-HCl (pH 7.5). Washed cells were suspended in 1 ml of the 
buffer solution described above and 5 .mu.l of a solution containing 200 
.mu.g of lysozyme and 0.25M EDTA was added. The mixture was centrifuged 
for 30 minutes to obtain a supernatant fluid. The amount of interferon in 
the supernatant was determined according to the method of Armstrong, 
wherein Sindvis virus was used as the virus and FL cells derived from 
human amnion cells were used as the animal cells. The results are shown in 
Table 3. 
TABLE 3 
______________________________________ 
Product encoded 
IFN-.gamma. 
Strains Plasmid by the plasmid 
(units/ml) 
______________________________________ 
IGBD1 pGBD1 IFN-.gamma. trace 
IGVL10 pGVL10 1-Ser--IFN-.gamma. 
8 .times. 10.sup.4 
IGVM101 pGVM101 3-Ser--IFN-.gamma. 
3 .times. 10.sup.5 
IGWE4 pGWE4 IFN-.gamma.(.DELTA.1-4) 
2 .times. 10.sup.5 
IGKA2 pGKA2 IFN-.gamma. 2 .times. 10.sup.4 
______________________________________ 
IGKA2 is a strain carrying plasmid pGKA2 coding for IFN-.gamma.. 
REFERENCE EXAMPLE 1 
Insertion of human IFN-.gamma. DNA into the expression vector pKYP-11: 
In this example, 6 .mu.g of plasmid pIFN.gamma.-G4 (3.6 Kb) was dissolved 
in 50 .mu.l (total volume) of a solution containing 20 mM Tris-HCl (pH 
7.5), 10 mM MgCl.sub.2, 10 mM dithiothreitol and 50 mM NaCl. Then, 12 
units each of restriction enzymes PvuII and HindIII were added and 
digestion reaction was carried out at 37.degree. C. for 4 hours. The 
reaction solution was heated at 65.degree. C. for 7 minutes to inactivate 
the enzymes and subjected to purification by LGT method to obtain 1.2 
.mu.g of a DNA fragment of 1.3 Kb containing human IFN-.gamma. DNA. 
Separately, 4 .mu.g of pKYP-11 was dissolved in 40 .mu.l (total volume) of 
a solution containing 20 mM Tris-HCl (pH 7.5), 10 mM MgCl.sub.2, 10 mM 
dithiothreitol and 50 mM NaCl. 8 units of BamHI was added and digestion 
reaction was carried out at 37.degree. C. for 3 hours. The reaction 
solution was heated at 65.degree. C. for 5 minutes to inactivate the 
enzyme. Thereafter, 30 .mu.M 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 .mu.l) was added. Fill-in reaction was carried 
out at 15.degree. C. for 1 hour and the reaction solution was heated at 
68.degree. C. for 15 minutes to inactivate DNA polymerase I. 10 units of 
HindIII was added and digestion reaction was carried out at 37.degree. C. 
for 3 hours, followed by heating at 65.degree. C. for 5 minutes to 
inactivate HindIII. The digestion reaction solution of the plasmid pKYP-11 
was subjected to purification by LGT method to obtain about 2.5 .mu.g of a 
DNA fragment of about 4.7 Kb containing p.sub.trp. 
Then, 0.5 .mu.g of the DNA fragment of 1.3 Kb containing human IFN-.gamma. 
DNA and 1.0 .mu.g of the DNA fragment of about 4.7 Kb containing 
P.sub.trp, which was obtained from the plasmid pKYP-11, were dissolved in 
20 .mu.l of a solution containing 20 mM Tris-HCl (pH 7.5), 6 mM 
MgCl.sub.2, 5 mM dithiothreitol and 500 .mu.M ATP, and 4 units of T4 DNA 
ligase (product of New England Biolabs) was added. Ligation reaction was 
carried out at 4.degree. C. for 18 hours, and Escherichia coli HB101 was 
transformed with the obtained recombinant plasmid mixture by conventional 
technique to obtain an Ap.sup.R colony. A plasmid, pGC-7 was separated 
from the culture broth of the colony. The structure of pGC-7 was confirmed 
by the digestion with HindIII, BamHI, HpaI, SalI, EcoRI and ClaI and 
agarose gel electrophoresis. Escherichia coli strain containing pGC-7 has 
been deposited with the FERM as Escherichia coli IGC-7 (FERM P-6814, FERM 
BP-497). 
REFERENCE EXAMPLE 2 
Construction of recombinant plasmid pGKA-2: 
In this example, 6 .mu.g of the pGC-7 DNA obtained in Reference Example 1 
was dissolved in 59 .mu.l (total volume) of a solution containing 20 mM 
Tris-HCl (pH 7.5), 10 mM MgCl.sub.2, 10 mM dithiothreitol and 10 mM NaCl, 
and 12 units of BstNI (product of New England Biolabs) was added. Reaction 
was carried out at 60.degree. C. for 3 hours, and the reaction solution 
was heated at 65.degree. C. for 5 minutes to inactivate BstNI. Then, 150 
mM NaCl and 8 units of SalI were added and digestion reaction was carried 
out at 37.degree. C. for 3 hours. The reaction solution was again heated 
at 65.degree. C. for 5 minutes to inactivate SalI and subjected to 
purification by LGT method to obtain about 0.8 .mu.g of a DNA fragment of 
about 1,125 bp containing the most part of the human IFN-.gamma. DNA. 
Separately, 3 .mu.g of pKYP-10 was dissolved in 40 .mu.l (total volume) of 
a solution containing 20 mM Tris-HCl (pH 7.5), 10 mM MgCl.sub.2, 10 mM 
dithiothreitol and 100 mM NaCl. 6 units each of HindIII and SalI were 
added and digestion reaction was carried out at 37.degree. C. for 3 hours. 
The reaction solution was heated at 65.degree. C. for 5 minutes to 
inactivate HindIII and SalI and subjected to purification by LGT method to 
obtain about 1.8 .mu.g of a DNA fragment of about 4.1 Kb containing 
P.sub.trp. 
The N-terminal amino acid of the mature human IFN-.gamma. polypeptide is 
Cys. In order to express mature IFN-.gamma. DNA, it is necessary to 
furnish an initiation codon (ATG) just before the 5'-terminal codon TGT 
(Cys) and further to adjust the length between SD-sequence downstream from 
Ptrp and ATG to a suitable length of 6-18 bp. Therefore, the following DNA 
linker was synthesized. 
##STR23## 
Two single chain DNAs of 18-mer and 15-mer were synthesized by a 
conventional triester method [R. Crea, et al.: Proc. Natl. Acad. Sci., USA 
75, 5765 (1978)]. Then, 2 .mu.g each of the 18-mer and 15-mer DNAs were 
dissolved in 20 .mu.l (total volume) of a solution containing 50 mM 
Tris-HCl (pH 7.5), 10 mM MgCl.sub.2, 5 mM dithiothreitol, 0.1 mM EDTA and 
1 mM ATP. 30 units of T4 polynucleotide kinase (product of Boehringer 
Mannheim GmbH) was added and phosphorylation reaction was carried out at 
37.degree. C. for 60 minutes. 
Then, 2 .mu.g each of phosphorylated 18-mer and 15-mer DNAs were mixed and 
the mixture was heated at 70.degree. C. for 5 minutes and allowed to stand 
at room temperature for annealing to obtain the DNA linker having the 
structure given above. 
0.4 .mu.g of the BstNI-SalI fragment of 1,125 bp obtained above and derived 
from pGC-7 and 1.0 .mu.g of the DNA fragment of 4.1 Kb obtained by 
digestion of the expression vector pKYP-10 with HindIII and SalI were 
dissolved in 25 .mu.l (total volume) of a solution containing 20 mM 
Tris-HCl (pH 7.5), 6 mM MgCl.sub.2, 5 mM dithiothreitol and 500 .mu.M ATP. 
About 0.1 .mu.g of the DNA linker mentioned above was added to the 
mixture, followed by addition of 6 units of T4 DNA ligase. Ligation 
reaction was carried out at 4.degree. C. for 17 hours. Escherichia coli 
HB101 was transformed using the obtained recombinant plasmid mixture by 
conventional technique to obtain an Ap.sup.R colony. A plasmid, pGKA-2 
illustrated in FIG. 5 was isolated from the culture broth of the colony. 
The structure of pGKA-2 was confirmed by the digestion with EcoRI, ClaI, 
HindIII, BstNI and SalI and agarose gel electrophoresis. It was confirmed 
by the method of Maxam-Gilbert that the base sequence from the SD-sequence 
(AAGG) to the initiation codon (ATG) in the plasmid pGKA-2 was 
"AAGGGTATCGATAAGCTTATG". 
Escherichia coli strain containing pGKA-2 has been deposited with the FERM 
as Escherichia coli IGKA-2 (FERM P-6798, FERM BP-496).