Highly purified protein, production and use thereof

A substantially pure non-glycosylated human interleukin-2 protein having a specific activity of not less than 10.sup.4 U/mg is obtained by growing a transformant carrying a DNA having a base sequence coding for human interleukin-2 to cause production and accumulation of human interleukin-2 in the culture broth, subjecting the thus obtained human interleukin-2-containing liquid to a purification process comprising a hydrophobic column chromatography.

This invention relates to a human interleukin-2 protein and a method of 
producing the same by a genetic engineering technique. 
Interleukin-2 [also called T cell growth factor (TCGF); hereinafter 
abbreviated as IL-2] is a lymphokine produced by T cells upon stimulation 
with a lectin or alloantigen, for example [Science, 193, 1007 (1976); 
Immunological Reviews, 51, 257 (1980)]. IL-2 facilitates long-period 
culture of T cells by enabling said cells grow in vitro while retaining 
their biological functions. Furthermore, IL-2 reportedly promotes the 
mitogen reaction of thymus cells (costimulator), restores the production 
by spleen cells of antibodies against T cell-dependent antigens in nude 
mice (T cell replacing factor) and/or promotes the differentiation and 
proliferation of killer cells (killer helper factor) [The Journal of 
Immunology, 123, 2928 (1979); Immunological Reviews, 51, 257 (1980)]. 
Killer T cell clones, helper T cell clones and, further, natural killer 
cell clones have been cloned through the utilization of IL-2 [e.g., 
Nature, 268, 154 (1977); The Journal of Immunology, 130, 981 (1983)]. In 
addition to such direct use in T cell or natural killer cell cloning, IL-2 
can be used in selectively growing killer T cells specific for certain 
antigens, for example killer T cells recognizing a tumor antigen and 
thereby destroying the tumor, in vitro. By injecting the tumor-specific 
killer T cells produced by use of IL-2 into an animal, it is possible to 
inhibit and prevent the tumor growth [The Journal of Immunology, 125, 1904 
(1980)]. It is also known that IL-2 induces the production of IFN-.gamma. 
[The Journal of Immunology, 130, 1784 (1983)] and that IL-2 activates 
natural killer cells [The Journal of Immunology, 130, 1970 (1983)]. 
The above experimental data suggest that IL-2 is useful as an antitumor 
agent. IL-2 is known to restore the helper T cell function in nude mice 
lacking thymic function [European Journal of Immunology, 10, 719 (1980)] 
or restore the induction of killer T cells against homologous cells 
[Nature, 284, 278 (1980)] and, therefore IL-2 is useful in treating 
immunocompromised diseases. 
Large scale production of IL-2 by methods known in the art has been 
unsuccessful. The development of clinical applications thereof has 
therefore been hindered by the scarcity and purity of IL-2. Accordingly, a 
technique for the production of highly purified IL-2 in large amounts, in 
an easy and simple manner and at low cost is highly desirable. 
Taniguchi et al. cloned a human IL-2 gene by using IL-2 mRNA isolated from 
the human T cell leukemia line Jurkat as the starting material, reported 
the amino acid sequence of human IL-2 protein as deduced therefrom and 
expressed the gene in COS-7 cells [Nature, 302, 305 (1983)]. Later, Devos 
et al. reported that a human spleen cell-derived IL-2 gene had been cloned 
and expressed in Escherichia coli [Nucleic Acids Research. 11, 4307 
(1983)]. Nevertheless, so far, the presence of an IL-2-like substance has 
only been estimated on the basis of detectable TCGF activity and there has 
been no report that a human IL-2 protein produced by a transformant 
carrying a DNA coding for human IL-2 has been isolated in a purified form. 
The present inventors have developed a technique by which substantially 
pure non-glycosylated IL-2 protein can be obtained through cloning a human 
IL-2 gene utilizing the gene manipulation technology, introduction of the 
recombinant DNA molecule obtained thereby into a host and expression of 
the human IL-2 gene in said host, and as a result could establish a method 
of producing a substantially pure, non-glycosylated human IL-2 protein and 
have completed the present invention. 
Thus, the present invention provides a substantially pure, non-glycosylated 
human IL-2 protein and a method of producing said human IL-2 protein which 
comprises cultivating a transformant carrying a DNA having a base sequence 
coding for human IL-2 and purifying said protein from the culture broth. 
DNA coding for human IL-2 to be used in the practice of the present 
invention is, for example, a DNA (I) having the base sequence defined by 
codons 1-133 in FIG. 2. This DNA (I), may have, at its 5' end, either ATG 
or a signal sequence represented in FIG. 2 by codons S1-S20 and, at its 3' 
end, preferably has TAA, TGA or TAG, more preferably TGA. 
The DNA (I) is preferably connected downstream from a promoter, for example 
the tryptophan (trp) promoter, rec A promoter or .lambda.PL promoter, more 
preferably the trp or .lambda.PL promoter. 
In accordance with the invention; the mRNA coding for human IL-2 is 
isolated from a culture of human peripheral leukocytes stimulated by 
concanavalin A, for instance, then a single-stranded cDNA is synthesized 
therefrom using reverse transcriptase, and further a double-stranded DNA 
is synthesized. Double-stranded DNA is inserted into a plasmid, the 
recombinant plasmid is used for transformation of a strain of Escherichia 
coli or Bacillus subtilis, for example, and the cDNA-containing plasmid is 
cloned. In this way, a double-stranded DNA coding for human IL-2 can be 
produced. 
More particularly, a human IL-2-encoding mRNA to be used in the practice of 
the invention can be obtained by the method of Hinuma et al. [Biochemical 
and Biophysical Research Communications, 109, 363 (1982)]. With the 
thus-obtained human IL-2 mRNA as a template, a cDNA is synthesized by the 
per se known method using reverse transcriptase, and the cDNA is converted 
to a double-stranded DNA [Maniatis, T. et al. Cell, 8, 163 (1976); Land, 
H. et al., Nucleic Acids Research, 9, 2251 (1981)]. The double-stranded 
DNA is inserted, for example, into the plasmid pBR322,at the PstI 
restriction endonuclease cleavage site by the dG-dC homopolymer ligation 
method [Nelson, T. S., Methods in Enzymology, 68, 41 (1979)], for 
instance. Furthermore, an oligonucleotide having a base sequence 
corresponding to the amino acid sequence of a part of human IL-2, for 
instance, is chemically synthesized and then labeled with .sup.32 P. Using 
this as a probe, a desired clone is selected from among tetracycline- or 
ampicillin-resistant transformants by the per se known colony 
hybridization method [Grunstein, M. and Hogness, D. S., Proc. Natl. Acad. 
Sci. USA, 72, 3961 (1975); Alwine, J. C. et al., Methods in Enzymology, 
68, 220 (1979)]. The presence of a human IL-2 gene is confirmed by 
determination of the base sequence of DNA derived from a clone selected by 
means of the above hybridization method by Maxam-Gilbert method [Maxam, A. 
M. et al., Proc. Natl. Acad. Sci. USA, 74, 560 (1977)] or the dinucleotide 
synthetic chain termination method using phage M13 [Messing, J. et al., 
Nucleic Acids Research, 9, 309 (1981)]. Then, a complete or partial human 
IL- 2 gene is excised from a selected clone obtained and inserted into a 
plasmid downstream from an appropriate promoter and the SD (Shine and 
Dalgarno) base sequence, for the subsequent introduction into an 
appropriate host. 
The use of the trp promoter, among others, as the promoter is preferred. As 
the host, a strain of Escherichia coli (e.g. strain 294, strain DH1, 
strain N4830), among others, is preferred. 
The strains Escherichia coli 294 [Beckman et al., Proc. Natl. Acad. Sci. 
USA, 73, 4174 (1976)], E. coli DH1 [Selson, M. E. et al., Nature, 217, 
1110 (1968)] and E. coli N4830 [Cell, 25, 713 (1981) ] are known available 
strains. 
The transformation of such hosts with the DNA is performed by the known 
method [Cohen, S. N. et al., Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)]. 
A transformed host thus obtained is cultivated in a per se known medium, 
such as glucose- and casamino acid-containing M9 medium [Miller, J. 
Experiments in Molecular Genetics, pages 431-433 (Cold Spring Harbor 
Laboratory, New York, 1972)]. In cases where the trp promoter is used, an 
agent such as 3-.beta.-indolylacrylic acid may be added for increasing the 
efficiency of said promoter. The cultivation is generally carried out at 
15.degree.-43.degree. C. for 10-30 hours, preferably 18-24 hours, with 
aeration and/or stirring as necessary. 
In a case where the .lambda.PL promoter is used, the cultivation is 
preferably carried out at a relatively low temperature of about 30.degree. 
to 36.degree. C. to grow the transformants and then of about 37.degree. to 
42.degree. C. to inactivate the repressor in the transformants and to 
effectively express the DNA coding for human IL-2. 
Human IL-2 thus formed may be assayed using an IL-2-dependent cell line. 
Since human IL-2 is known to promote the growth of rat, mouse or other 
IL-2-dependent cells as well as human IL-2-dependent cells [Immunological 
Reviews, 51, 257 (1980)], not only IL-2-dependent human cell lines but 
also rat or mouse IL-2-dependent cell lines may be used for the assay 
[Journal of Immunology, 130, 981 and 988 (1983)]. The thus obtained IL-2 
can be used to maintain desired cell lines which are IL-2 dependent. 
In particular, mouse IL-2-dependent cell lines may be maintained stably by 
subculturing for a long period of time, so that their use can give highly 
reproducible assay results. 
In extracting human IL-2 according to the invention from cultivated cells, 
the cells, after cultivation, are harvested by known methods, suspended 
either in a solution of a protein denaturing agent such as a mineral acid 
salt of guanidine (e.g. hydrochloride, nitrate, sulfate), and the 
suspension is stirred in the cold and then centrifuged, whereby an 
IL-2-containing supernatant is obtained, or in a buffer and disrupted by 
sonication, lysozyme treatment and/or freezing and thawing, followed by 
centrifugation to give an IL-2-containing supernatant. Any other method 
which is useful to extract IL-2 may also be used. 
The isolation of IL-2 from the above supernatant and the subsequent 
purification of the same can be carried out by an appropriate combination 
of per se known methods of separation and purification. Such known 
separation and purification methods include, among others, methods 
utilizing solubility differences such as salting out methods and solvent 
precipitation methods, methods utilizing molecular weight differences in 
the main, such as dialysis, ultrafiltration, gel filtration and 
SDS-polyacrylamide gel electrophoresis, methods utilizing charge 
differences, such as ion exchange chromatography, methods utilizing 
specific affinities such as affinity chromatography, methods utilizing 
hydrophobicity differences, such as reversed phase high performance liquid 
chromatography, and methods utilizing isoelectric point differences, such 
as isoelectric focusing. Since human IL-2 protein is highly hydrophobic, 
hydrophobic column chromatography, in particular high performance liquid 
chromatography using a reversed phase column, is preferred in purifying 
said protein. 
Thus, for example, the cells obtained by cultivating a strain of 
Escherichia coli carrying a gene coding for human IL-2 are suspended in a 
solution, preferably in a buffer of a protein denaturing agent such as 
guanidine hydrochloride(in a concentration of 2M to 8M, preferably 6M-8M), 
and the suspension is stirred, preferably at 0.degree.-5.degree. C. for 
0.5-3 hours and then centrifuged. The supernatant thus collected is, if 
appropriate, concentrated using an ultrafiltration device or the like and 
subjected to dialysis. The resulting precipitate, if any, is removed by 
centrifugation and the supernatant is subjected to anion exchange 
chromatography using diethylaminoethylcellulose [e.g. DE 52 cellulose 
(Whatman, Gt. Britain) column], for example, and an active, i.e. having 
IL-2 activity, fraction is collected. 
Then, after concentration using an ultrafiltration device, the active 
fraction is subjected to gel filtration using 
N,N'-methylenebisacrylamide-crosslinked allyldextran, such as a Sephacryl 
S-200 (Pharmacia, Sweden) column, or the like. The active fraction 
collected is then subjected to the above-mentioned high performance liquid 
chromatography. In this manner, non-glycosylated human IL-2 according to 
the invention can be obtained. 
As the reversed phase system to be used in high Performance liquid 
chromatography, resin coated with alkylated (C.sub.1-18) silanes are 
effectively used among others. As the eluent, a C.sub.1-6 lower alkanol 
(e.g. ethanol, propanol) or acetonitrile can advantageously be used and 
the eluent has a pH of 1.5-8, preferably 1.5-4. The rate of elution is 
0.1-100 ml/min, preferably 0.5-10 ml/min. 
Human IL-2 protein thus obtained can be turned into a powder by 
lyophilization as necessary. On the occasion of lyophilization, a 
stabilizer such as sorbitol, mannitol, dextrose, maltose, glycerol or 
human serum albumin (HSA) may be added. 
When assayed for IL-2 activity using, as the index, the radioactive 
thymidine uptake by IL-2-dependent mouse cells, human IL-2 protein 
obtained in accordance with the present invention shows a specific 
activity of not less than 1.times.10.sup.4 U/mg. In accordance with the 
invention, there can be obtained a highly pure, non-glycosylated human 
IL-2 protein having a specific activity of not less than 10.sup.4 U/mg, 
and preferably about 2.times.10.sup.4 U/mg to 4.times.10.sup.4 U/mg. 
As mentioned herein, the IL-2 activity in units (U) may be calculated in 
the following manner. 
Thus, an IL-2-containing sample is added to a medium containing cells of a 
mouse cell line which grow depending on the concentration of IL-2, and the 
whole mixture is incubated at 37.degree. C. overnight in the presence of 
5% CO.sub.2, the growth of said-cell line may be determined using 
tritiated thymidine. For calculating the IL-2 activity in units (U) in the 
sample in question, a standard IL-2 (1 U/ml) is used for comparison and 
said activity in units is calculated from the activity ratio between the 
sample and the standard. 
More particularly, cells of an IL-2-dependent mouse cell line (NKC3) 
[Hinuma et al., Biochem. Biophys. Res. Commun., 109, 363 (1982)] 
maintained by subculturing in 20% FCS-added RPMI 1640 medium supplemented 
with an IL-2-containing conditioned medium at 37.degree. C. in the 
presence of 5% CO.sub.2 are washed twice with a serum-free RPMI 1640 
medium and resuspended in 20% FCS-added RPMI 1640 medium to 
6.times.10.sup.5 cells/ml. 
An IL-2-containing sample is distributed, in 50-.mu.l portions, into the 
wells in the first row of a 96-well flat-bottomed microtiter plate (Nunc, 
Denmark). Using 50-.mu.l portions of 20% FCS-added RPMI 1640 medium, a two 
fold serial dilution is produced until the 12th row. Then the 
above-mentioned NKC3 cell suspension is distributed, in 50-.mu.l portions, 
into all the wells, followed by incubation at 37.degree. C. in the 
presence of 5% CO.sub.2 for 24 hours. After 20 hours of incubation, 1 
.mu.Ci of tritiated thymidine (Amersham, Great Britain) is added to each 
well. After continued incubation for 4 hours, cells are recovered onto a 
glass filter using a cell harvester (Flow, USA) and measured for tritiated 
thymidine uptake using a liquid scintillation counter. In carrying out the 
above assay, a standard IL-2 sample is subjected to the same procedures as 
an IL-2 containing sample to be assayed and the tritiated thymidine uptake 
is determined. 
The calculation of the activity in units (U) is carried out by the probit 
method according to the Journal of Immunology, 120, 2027 (1978). Thus, the 
maximum uptake in the standard IL-2 sample dilution series is regarded as 
100%, and the percentage (%) of the uptake for each dilution stage is 
calculated. The values thus obtained are plotted on a normal probability 
paper and the dilution factor at which the uptake is 50% is determined 
graphically. Also for each IL-2-containing sample, the dilution factor at 
which the uptake is 50% is determined in the same manner. The amount of 
IL-2 activity contained in the culture supernatant after 48 hours of 
incubation, at 37.degree. C. in the presence of 5% CO.sub.2, of a 
suspension of human peripheral blood lymphocytes (5.times.10.sup.6 
cells/ml) in 10% FCS-added RPMI 1640 medium supplemented with 40 .mu.g/ml 
of concanavalin A and 15 ng/ml of 12-O-tetradecanoylphorbol-13-acetate is 
defined as 1 U/ml. 
The IL-2 concentration (U/ml) of the sample is calculated by the formula: 
##EQU1## 
The natural IL-2 obtained from the human peripheral blood had a specific 
activity of 20,000-70,000 U/mg as determined by the above assay method. 
This activity is almost equal to the activity of non-glycosylated human 
IL-2 protein according to the present invention. 
Non-glycosylated human IL-2 protein according to the invention preferably 
comprises the polypeptide (II) having the amino acid sequence shown in 
FIG. 3 wherein X is Met or hydrogen. 
Human IL-2 protein produced in accordance with the invention has the 
following characteristics: 
1) It is homogeneous in SDS-polyacrylamide gel electrophoresis and has a 
molecular weight of 15,000.+-.1,000 daltons as determined by said 
electrophoresis; 
2) It contains alanine or methionine as the amino-terminal amino acid; 
3) It contains threonine as the carboxy-terminal amino acid; 
4) It promotes growth of T cells or natural killer cells while maintaining 
their functions. 
Human IL-2 protein produced in accordance with the invention reacts 
negatively in the limulus lysate test [Haemostasis, 7, 183 (1978)] and is 
low in protein impurities and pyrogen content, so that it can be used 
safely as a bulk substance for manufacturing injections and so on. 
Non-glycosylated human IL-2 protein obtained in accordance with the present 
invention has an activity of promoting the growth of normal T cells or 
natural killer cells while maintaining their functions. Therefore, IL-2 
protein according to the invention can be used in growing and subculturing 
T cells or natural killer cells in vitro for a long period of time or 
cloning the same. Moreover, this property can be utilized in human IL-2 
activity measurement. 
Furthermore, human IL-2 protein of the invention makes it possible to 
selectively grow antigen-specific killer T cells, which recognize and 
destroy tumor antigens, or natural killer cells, which are capable of 
killing tumor cells irrespective of the presence or absence of an 
experience of antigenic sensitization, in vitro. When inoculated into a 
living organism simultaneously with the introduction of said killer cells 
into the living organism, human IL-2 of the present invention increases 
the anti-tumor effete of killer T cells. Therefore, said IL-2 is useful 
for the prevention or treatment of tumors or the treatment of 
immunodeficiency diseases in warm-blooded mammals (e.g. mouse, rat, 
rabbit, dog, cat, pig, horse, sheep, cattle, human). 
Human IL-2 protein according to the present invention is a highly purified 
product and has little antigenicity for humans and is low in toxicity. 
As an agent for the prevention and treatment of tumors, human IL-2 protein 
of the invention can be administered either parenterally or orally in the 
form of, for example, injections or capsules prepared by appropriate 
blending or dilution with a per se known carrier. It can be used either 
alone or in combination with killer T cells or natural killer cells grown 
in vitro, as mentioned hereinbefore. 
Human IL-2 protein according to the present invention has substantially the 
same biological activity as the natural human IL-2 and accordingly can be 
used in the same manner as said natural IL-2. The dissociation constant of 
IL-2 in relation to the IL-2 receptor of the responding cells is very 
small, so that a very small dose is sufficient in most cases. 
For the purpose of growing T cells in vitro, the human IL-2 of the 
invention can be added to the medium in a concentration of about 0.01-1 
U/ml, preferably about 0.1-0.5 U/ml. 
In an example of the use for the purpose of growing T cells in vitro, IL-2 
protein of the invention is added, at a concentration of 0.1-0.5 unit/ml, 
to a cell suspension containing, for example, alloantigen-sensitized T 
cells obtained by 3-day mixed lymphocyte culture of human peripheral 
blood-derived T cells (1.times.10.sup.6 cells/ml), with B cell 
transformants (1.times.10.sup.6 cells/ml) resulting from X ray irradiation 
(1,500 rads) added, in RPMI 1640 medium containing 20% of fetal bovine 
serum. Cultivation is continued for about 1 month while repeating medium 
exchange an about one-week intervals. 
The transformant disclosed in the following examples, namely Escherichia 
coli DH1/pTF4, has been deposited at Institute for Fermentation, Osaka 
(IFO) under the deposit number IFO-14299, and also deposited at 
Fermentation Research Institute, Agency of Industrial Science and 
Technology, Ministry of International Trade and Industry, Japan (FRI) 
under the accession number of FERM BP-628 of a deposit under the Budapest 
Treaty. 
In the present specification and drawings, the bases and amino acids, when 
indicated by abbreviations, are abbreviated according to the rules of the 
IU-IUB Commission on Biochemical Nomenclature or the practice in the 
field concerned. Examples are shown in Table 1. Where optical isomerism is 
involved, the amino acids mentioned are in the L form unless otherwise 
specifically indicated. 
TABLE 1 
______________________________________ 
DNA: Deoxyribonucleic acid 
CDNA: Complementary deoxyribonucleic acid 
A: Adenine 
T: Thymine 
G: Guanine 
C: Cytosine 
RNA: Ribonucleic acid 
mRNA: Messenger ribonucleic acid 
DATP: Deoxyadenosine triphosphate 
DTTP: Deoxythymidine triphosphate 
dGTP: Deoxyguanosine triphosphate 
dCTP: Deoxycytidine triphosphate 
ATP: Adenosine triphosphate 
EDTA: Ethylenediaminetetraacetic acid 
SDS: Sodium dodecyl sulfate 
Gly: Glycine 
Ala: Alanine 
Val: Valine 
Leu: Leucine 
Ile: Isoleucine 
Ser: Serine 
Thr: Threonine 
Cys: Cysterine 
1/2 Cys: Half cystine 
Met: Methionine 
Glu: Glutamic acid 
Asp: Aspartic acid 
Lys: Lysine 
Arg: Arginine 
His: Histidine 
Phe: Phenylalanine 
Tyr: Tyrosine 
Trp: Tryptophan 
Pro: Proline 
Asn: Asparagine 
Gln: Glutamine 
______________________________________

REFERENCE EXAMPLE 1 
(i) Isolation of mRNA coding for human IL-2 
Lymphocytes prepared from human peripheral blood were incubated in RPMI 
1640 medium supplemented with 10% fetal bovine serum, 
12-O-tetradecanoylphorbol-13-acetate (TPA) (15 ng/nl) and concanavalin A 
(40 .mu.g/ml) at 37.degree. C. to thereby induce production of IL-2. After 
24 hours of incubation, 1.times.10.sup.10 human lymphocytes thus induced 
were disrupted and denatured in a solution containing 5M guanidine 
thiocyanate, 5% mercaptoethanol, 50 mM Tris.multidot.HCl (pH 7.6) and 10 
mM EDTA using a Teflon homogenizer, then sodium N-lauroylsarcosinate was 
added to a concentration of 4%, and the mixture, after homogenization, was 
layered onto 6 ml of 5.7M cesium chloride solution (5.7M cesium chloride, 
1M EDTA) and centrifuged using a Beckman SW28 rotor at 24,000 rpm and 
15.degree. C. for 48 hours, to give an RNA precipitate. This RNA 
precipitate was dissolved in 0.25% sodium N-lauroylsarcosinate and 
precipitated with ethanol to give 10 mg of RNA. In a high-concentration 
salt solution (0.5M NaCl, 10 mM Tris.multidot.HCl pH 7.6, 1 mM EDTA, 0.3% 
SDS), this RNA was adsorbed on an oligo(dT)cellulose column. Elution with 
a low-concentration salt solution (10 mM Tris.multidot.HCl pH 7.6, 1 mM 
EDTA, 0.3% SDS) gave 300 .mu.g of poly(A)-containing mRNA. This mRNA was 
further subjected to precipitation with ethanol, then dissolved in 0.2 ml 
of a solution (10 mM Tris.multidot.HCl pH 7.6, 2mM EDTA, 0.3% SDS), 
treated at 65.degree. C. for 2 minutes and fractionated by 10-35% sucrose 
density gradient centrifugation (at 20.degree. C. and 25,000 rpm for 21 
hours using a Beckman SW28 rotor) into 22 fractions. An aliquot of each 
fraction was injected into oocytes of Xenopus laevis and the IL-2 activity 
in proteins thus synthesized was measured. Fractions Nos. 11-15 
(sedimentation coefficient 8S-15S) were found to have IL-2 activity. About 
25 .mu.g of IL-2 mRNA was contained in these fractions. 
(ii) Synthesis of single-stranded DNA 
Using the mRNA obtained above and reverse transcriptase, synthesis of 
single stranded cDNA was carried out in 100 .mu.l of reaction solution (5 
.mu.g of mRNA, 50 .mu.g of oligo(dT), 100 units of reverse transcriptase, 
1 mM dATP, 1 mM dCTP, 1 mM dGTP, 1 mM dTTP, 8 mM MgCl.sub.2, 50 mM KCl, 10 
mM dithiothreitol, 50 mM Tris.multidot.HCl pH 8.3) at 42.degree. C. for 1 
hour, followed by deproteinization with phenol and treatment with 0.1N 
NaOH at 70.degree. C. for 20 minutes for removal of RNA by decomposition. 
(iii) Synthesis of double-stranded DNA 
Double-stranded DNA encoding IL-2 was synthesized by treating the 
thus-synthesized single-stranded complementary DNA obtained in (ii) above 
in 50 .mu.l of a reaction solution (the same as the above-mentioned 
reaction solution of step (ii) except that mRNA and oligo(dT) were 
absenct) at 42.degree. C. for 2 hours. See Maniatis et al. supra. 
(iv) Addition of dC tail 
Double-stranded DNA from step(iii) was treated with nuclease Sl in 50 .mu.l 
of a reaction solution (double-stranded DNA, 0.1M sodium acetate pH 4.5, 
0.25M NaCl, 1.5 mM ZnSO.sub.4, 60 units of Sl nuclease) at room 
temperature for 30 minutes, followed by deproteinization with phenol and 
DNA precipitation with ethanol. The DNA precipitate was treated with 
terminal transferase in 50 .mu.l of a reaction solution (double-stranded 
DNA, 0.14M potassium cacodylate, 0.3M Tris (base) pH 7.6, 2 mM 
dithiothreitol, 1 mM CoCl.sub.2, 0.15 mM dCTP, 30 units of terminal 
transferase) at 37.degree. C. for 3 minutes to thereby cause extension of 
the double-stranded DNA by about 15 deoxycytidines at both the 3' ends. 
This series of reactions gave about 300 ng of a deoxycytidine 
chain-containing double-stranded DNA. 
(v) Cleavage of Escherichia coli plasmid and addition of dG tail 
Separately, 10 .mu.g of Escherichia coli plasmid pBR322 DNA was treated 
with the restriction enzyme PstI in 50 .mu.l of a reaction solution (10 
.mu.g of said DNA, 50 mM NaCl, 6 mM Tris.multidot.HCl pH 7.4, 6 mM 
MgCl.sub.2, 6 mM 2-mercaptoethanol, 100 .mu.g/ml bovine serum albumin, 20 
units of PstI) at 37.degree. C. for 3 hours to thereby cleave the one PstI 
recognition site occurring in the pBR322 DNA, followed by deproteinization 
with phenol. The cleaved plasmid pBR322 DNA was extended by about 17 
deoxyguanines at both of its 3' ends by treating said DNA with terminal 
transferase in 50 .mu.l of a reaction solution (10 .mu.g of DNA, 0.14M 
potassium cacodylate, 0.3M Tris base pH 7.6, 2 mM dithiothreitol, 1 mM 
CoCl.sub.2, 0.15 mM GTP and 30 units of terminal transferase) at 
37.degree. C. for 3 minutes. 
(vi) Annealing of cDNA and transformation of Escherichia coli 
DNA coding for IL-2 obtained in (iv) above (0.1 .mu.g) and 0.5 .mu.g of 
plasmid DNA from (v) above were annealed by heating in a solution 
containing 0.1M NaCl, 50 mM Tris.multidot.HCl pH 7.6 and 1 mM EDTA at 
65.degree. C. for 2 minutes and then at 45.degree. C. for 2 hours followed 
by gradual cooling and the product was used for transformation of 
Escherichia coli MM294 in accordance with the method of Enea et al. [J. 
Mol. Biol., 96, 495 (1975)]. 
(vii) Isolation of cDNA-containing plasmid 
In this way, about 20,000 tetracycline-resistant transformants were 
isolated. DNAs of each of them were fixed on a nitrocellulose filter. 
Based on the amino acid sequence of IL-2 reported by Taniguchi et al. 
[Nature, 302, 305 (1983)], the complementary oligonucleotides of base 
sequences (.sup.5' AAA CAT CTT CAG TGT.sup.3' and .sup.5' ACA TTC ATG TGT 
GAA.sup.3') correponding to amino acids Nos. 74-78 (Lys-His-Leu-Gln-Cys) 
and amino acids Nos, 122-126 (Thr-Phe-Met-Cys-Glu), respectively, were 
chemically synthesized by the phosphotriester method [Crea, R. et al., 
Proc. Natl. Acad. Sci. USA, 75, 5765 (1978)]. 
These oligonucleotides were labeled with .sup.32 p at the 5' end by 
treatment with T4 polynucleotide kinase in 50 .mu.l of a reaction solution 
(0.20 .mu.g of oligonucleotide, 50 mM Tris.multidot.HCl pH 8.0, 10 mM 
MgCl.sub.2, 10 mM 2-mercaptoethanol, 50 .mu.Ci of .gamma.-.sup.32 p ATP, 3 
units of T4 polynucleotide kinase) at 37.degree. C. for 1 hour. These 
labeled oligonucleotides, as probes, were annealed with the 
above-mentioned DNAs fixed on nitrocellulose filter by the method of Lawn 
et al. [Nucleic Acids Res., 9, 6103 (1981)]. Four transformants reactive 
to the above two oligonucleotide probes were detected by autoradiography. 
Plasmid DNA was isolated from cells of each of these transformants by the 
Birnboim-Doly alkali method [Birnboim, H. C. & Doly, J., Nucleic Acids 
Res., 7, 1513 (1979)]. Then the insert in the plasmid DNA was cut out 
using the restriction enzyme PstI. From among the plasmids isolated, the 
one containing the longest insert encoding IL-2 was selected and named 
pILOT 135-8. The restriction enzyme map of this plasmid is shown in FIG. 
1. 
The primary structure (base sequence) of the cDNA inserted in this pILOT 
135-8 was determined by the dideoxynucleotide method and the Maxam-Gilbert 
method. The primary structure thus determined is shown in FIG. 2. The 
peptide defined by this base sequence consists of 153 amino acids, 
starting with the synthesis start signal (Nos. 64-66 ATG). Of these, the 
20 amino acids from the N-terminal are considered to constitute a signal 
peptide. The above primary structure has revealed that pILOT 135-8 has a 
base sequence coding for human IL-2 protein. This fact indicates that 
insertion of a gene coding for IL-2 obtained in (iv) above into an 
appropriate expression plasmid can lead to production of the IL-2 protein. 
REFERENCE EXAMPLE 2 
The plasmid pILOT135-8 obtained in Reference Example 1 was cleaved with the 
restriction enzyme HgiAl. The thus-obtained 1294 bp DNA fragment 
containing an IL-2 gene was treated with T4 DNA polymerase, joined with 
the ClaI linker CGATA ATG GCA, which contained the codon GCA for alanine 
and the codon ATG for methionine, and the product was treated with Clal 
and PstI, followed by insertion into ptrp771 at the ClaI-PstI site. The 
expression plasmid thus obtained was named pTF4 (FIG. 4). 
REFERENCE EXAMPLE 3 
Escherichia coli DH1 was transfomed with the plasmid pTF4 obtained in 
Reference Example 2 in accordance with the method of Cohen et al. [Proc. 
Natl. Acad. Sci. USA, 69, 2110 (1972)] to obtain a transformant 
(Escherichia coli DH1/pTF4 ) carrying said plasmid. 
EXAMPLE 1 
(i) E. coli DH1/pTF4 (obtained in Reference Example 3) was inoculated into 
50 ml of a liquid medium (pH 7.0) containing 1% Bacto tryptone (Difco 
Laboratories, USA), 0.5% Bacto yeast extract (Difco Laboratories, USA), 
0.5% sodium chloride and 7 .mu.g/ml tetracycline and placed in a 250-ml 
erlenmeyer flask. After incubation at 37.degree. C. overnight on a swing 
rotor, the culture medium was transferred to a 5-liter jar fermenter 
containing 2.5 liters of M9 medium containing 0.5% casamino acids, 0.5% 
glucose and 7 .mu.g/ml tetracycline. Incubation was then conducted with 
aeration and stirring at 37.degree. C. for 4 hours and, after addition of 
3-.beta.-indolylacrylic acid (25 .mu.g/ml), for an additional 4 hours. 
Cells were harvested from the thus-obtained 2.5-liter culture broth by 
centrifugation, frozen at -80.degree. C. and stored. 
(ii) The freeze-stored cells (12.1 g) obtained in Example 1(i) were 
suspended in 100 ml of an extractant (pH 7.0) containing 7M guanidine 
hydrochloride and 0.1M Tris.multidot.HCl, the suspension was stirred at 
4.degree. C. for 1 hour and the lysate was centrifuged at 28,000.times.g 
for 20 minutes to obtain 93 mi of a supernatant. 
(iii) Separately, various procedures were conducted to extract IL-2 from 
the transformant E. coli DH1/pTF4 cells obtained by the method of Example 
1 (i) to compare the respective extraction efficiencies. 
In the lysozyme-EDTA method, 2 g of E. coli DH1/pTF4 cells obtained in 
Example 1(i) were mixed with 16 ml of solution (pH7.0) containing 0.1M 
Tris-HCl, 10 mM EDTA and 250 mg/l lysozyme, the mixture was stirred at 
4.degree. C. for 1 hour and subseqently at 37.degree. C. for 5 minutes and 
the lysate was centrifuged at 28,000.times.g for 20 minutes. 
In the sonication method, 2 g of the cells from Example 1(i) were suspended 
in 16 ml of the solution (pH7.0) containing 0.1M Tris-HCl, the suspension 
was subjected to sonication at 0.degree. C. for 5 minutes and the lysate 
was centrifuged at 28,000.times.g for 20 minutes. 
In the guanidine-HCl method, 2 g of the cells from Example 1(i) were mixed 
with 16 ml of the solutions (pH7.0) containing 0.1M Tris-HCl and 2M, 4M or 
7M guanidine-HCl, the mixtures were stirred at 4.degree. C. for 1 hour and 
the lysates were centrifuged at 28,000.times.g for 20 minutes. The 
supernatant fluids thus obtained were used for the measurements of protein 
concentration and IL-2 activity. 
The results are summarized in Table 2. 
TABLE 2 
______________________________________ 
Extraction of IL-2 
Protein IL-2 activity 
Relative 
Extraction concentration 
in the extract 
ratio 
procedure (mg/ml) (U/ml) (%) 
______________________________________ 
Lysozyme-EDTA 
5.40 3.3 0.02 
Sonication 6.54 2.2 0.01 
2M Gu.HCl 2.60 46 0.2 
4M Gu.HCl 4.12 144 0.8 
7M Gu.HCl 7.22 19100 100 
______________________________________ 
Gu.HCl: guanidine hydrochloride 
(iv) The supernatant fluid obtained in Example 1(ii) was dialyzed against 
0.01M Tris.multidot.HCl buffer (pH 8.5) and then centrifuged at 
19,000.times.g for 10 minutes to give 94 ml of a dialyzed supernatant 
fluid. This was applied to a DE 52 (DEAE-cellulose, Whatman, Great 
Britain) column (50 ml in volume) equilibrated with 0.01M 
Tris.multidot.HCl buffer (pH 8.5) for protein adsorption. Proteins were 
eluted with a linear NaCl concentration gradient (0-0.15M NaCl, 1 liter). 
The fractions with IL-2 activity (53 ml) were concentrated to 4.8 ml using 
a YM-5 membrane (Amicon, USA) and subjected to gel filtration using a 
Sephacryl S-200 (Pharmacia, Sweden) column (500 ml in volume) equibrated 
with 0.1M Tris.multidot.HCl (pH 8.0)-1M NaCl buffer. The active fractions 
(28 ml) thus obtained were concentrated to 2.5 ml using a YM-5 membrane. 
The concentrate was applied to an Ultrapore RPSC (Altex, USA) column for 
adsoprtion, and high performance liquid chromatography was performed using 
a trifluoroacetic acid-acetonitrile system as the mobile phase. 
The conditions used: column, Ultrapore RPSC (4.6.times.75 mm); column 
temperature, 30.degree. C.; solvent A, 0.1% trifluoroacetic acid-99.9% 
water; solvent B, 0.1% trifluoroacetic acid-99.9% acetonitrile; elution 
program, minute 0 (68% A+32% B)-minute 25 (55% A+45% B)-minute 35 (45% 
A+55% B)-minute 45 (30% A+70% B)-minute 48 (100% B); elution rate, 0.8 
ml/min; detection wave length, 230 nm. An active fraction was collected at 
a retention time of about 39 minutes. Thus was obtained 10 ml of a 
solution containing 0.53 mg of non-glycosylated human IL-2 protein 
[specific activity, 30,000 U/mg; activity recovery from the starting 
material, 30.6%; purity of protein, 99% (determined by densitometry on an 
SDS-polyacrylamide gel electrophoretogram)]. 
Lyophilization of the above solution gave a white powder. The powder had a 
specific activity of 26,000 U/mg. 
(V) Human IL-2 protein obtained in Example 1 (iv) was examined for the 
following properties: 
(1) Homogeneity: 
Staining with Coomassie Brilliant Blue following SDS-polyacrylamide gel 
electrophoresis according to Laemmli et al. [Nature, 227, 680 (1970)] 
revealed only a single band of said human IL-2 protein (cf. FIG. 5). The 
location of the band remained unchanged under reducing conditions as well 
as under non-reducing conditions. 
(2) Molecular weight: 
The molecular weight of said human IL-2 protein was calculated to be about 
15,000 daltons based on the result of SDS-polyacrylamide gel 
electrophoresis (cf. FIG. 5). 
(3) Amino acid composition: 
A 20-.mu.g portion of said human IL-2 protein was placed in a glass test 
tube for hydrolysis, constant boiling hydrochloric acid containing 4% 
thioglycolic acid was added, the tube was then sealed in vacuo and 
hydrolysis was performed at 110.degree. C. for 24, 48 or 72 hours. After 
hydrolysis, the tube was opened, the hydrochloric acid was removed under 
reduced pressure, and the residue was dissolved in 0.02N hydrochloric acid 
and subjected to amino acid analysis using a Hitachi model 835 amino acid 
analyzer. For determination of cystine and cysteine, said human IL-2 
protein was oxidized with performic acid by the method of Hirs [Methods in 
Enzymol., 11, 197 (1967)] followed by hydrolysis in constant boiling 
hydrochloric acid under reduced pressure for 24 hours. The hydrolyzate was 
subjected to cysteic acid determination using an amino acid analyzer. The 
results obtained by the above amino acid analyses are shown in Table 3. 
The values are each the mean of three values respectively obtained after 
24, 48 and 72 hours of hydrolysis except for the values for serine and 
threonine which were determined by extrapolation to zero time of 
hydrolysis. 
TABLE 3 
______________________________________ 
Amino acid 
Mole % 
______________________________________ 
Asp/Asn 8.8 
Thr 9.3 
Ser 5.7 
Glu/Gln 13.7 
Pro 3.4 
Gly 1.7 
Ala 3.8 
1/2 Cys 2.3 
Val 3.1 
Met 3.7 
Ile 6.3 
Leu 16.3 
Tyr 2.3 
Phe 4.5 
Lys 8.3 
His 2.5 
Arg 3.1 
Trp 1.1 
______________________________________ 
(4) N-Terminal amino acid sequence: 
A 34-.mu.g portion of said human Il-2 protein was analyzed for the 
N-terminal amino acid sequence by applying the automatic Edman degradation 
method using a gas-phase protein sequenator (model 470A, Applied 
Biosystems, USA). Phenylthiohydantoin-amino acids (PTH-amino acids) were 
identified by high performance liquid chromatography using a Micropak-SP 
column (Varian, USA). The PTH-amino acid or acids detected in each step 
are shown in Table 4. 
TABLE 4 
______________________________________ 
Step PTH-amino acid detected 
______________________________________ 
1 Ala 
Met 
2 Pro 
Ala 
3 Thr 
Pro 
4 Ser 
Thr 
5 Ser 
6 Ser 
7 Thr 
8 Lys 
9 Lys 
10 Thr 
11 Gln 
12 Leu 
13 Gln 
14 Leu 
15 Glu 
16 Y* 
17 Leu 
18 Leu 
19 Leu 
20 Asp 
______________________________________ 
*Not yet identified. 
(5) C-Terminal amino acid: 
A 33-.mu.g portion of said human IL-2 protein was placed in a glass test 
tube for hydrazine degradation, 0.05 ml of anhydrous hydrazine was added 
and the tube was sealed in vacuo and heated at 100.degree. C. for 6 hours. 
The hydrazine degradation product obtained was lyophilized and dissolved 
in distilled water. Benzaldehyde was added to the solution, the mixture 
was stirred at room temperature for 1 hour and then centrifuged. The 
aqueous layer thus obtained was lyophilized and subjected to amino acid 
analysis using a Hitachi model 835 amino acid analyzer. Threonine alone 
was detected. 
(6) Tryptic peptide mapping 
A 15-.mu.g portion of said human IL-2 protein was digested with 0.4 .mu.g 
of trypsin (Washington, USA) in 120 .mu.l of 0.02M sodium hydrogen 
carbonate at 37.degree. C. for 18 hours. Then, 5 .mu.l of 
2-mercaptoethanol was added and the reaction was continued at 37.degree. 
C. for an additional 2 hours. Thereafter, the reaction was terminated by 
addition of 75 .mu.l of 1% trifluoroacetic acid. High performance liquid 
chromatography of the reaction mixture, which was performed under the 
conditions given below, gave an elution pattern shown in FIG. 6. 
Column: Ultrasphere-Octyl (5 .mu.m, 4.6.times.250 mm; Altex, USA); 
Column temperature: 30.degree. C.; 
Mobile phase: 
Solvent A, 0.02% trifluoroacetic acid-99.98% water; 
Solvent B, 0.02% trifuloroacetic acid-99.98% acetonitrile; 
Minute 0 (95% solvent A+5% solvent B)-minute 40 (30% solvent A+70% solvent 
B); 
Flow rate: 1.0 ml/minute; 
Detection: Fluorescence method [Analytical Biochem., 67, 438 (1975)] using 
fluorescamine (Roche, USA). 
(7) Activity against IL-2-dependent cell lines 
Assay of non-glycosylated human IL-2 protein according to the invention in 
accordance with the method described in Biochem. Biophys. Res. Commun., 
109, 363 (1982) revealed that said IL-2 protein had an activity that 
promoted tritiated thymidine uptake in an IL-2-dependent mouse cell line 
(NKC3; cf. FIG. 7) as well as in an IL-2-dependent human cell line (cf. 
FIG. 8). 
Furthermore, said IL-2 protein was dissolved in 20% FCS-added RPMI-1640 
medium to a concentration of 0.5 U/ml, and 2.times.10.sup.5 cells/ml of 
the NKC3 cell line were suspended in the medium. Subculturing was 
continued on a Linbro Multi dish (Flow, USA) at 37.degree. C. in the 
presence of 5% CO.sub.2 while repeating viable cell counting and 
resuspending the culture in a new fresh medium at 2- or 3-day intervals. 
As a result, said IL-2 protein was found to have an activity to maintain 
the growth of the NKC3 cell line for a long period of time, as illustrated 
in FIG. 9. 
EXAMPLE 2 
(Preparation for injection) 
The non-glycosylated human IL-2 protein-containing solution obtained in 
Example 1 (iv) is applied to a CM Toyopearl (Toyo Soda, Japan) column 
equilibrated with 0.025M ammonium acetate buffer (pH 5.0) under aseptic 
conditions for adsorption, followed by elution with the above buffer 
containing 0.15M NaCl. The eluate is diluted by addition of an appropriate 
amount of 0.15M NaCl, then HSA is added to a concentration of 0.5%, and 
the mixture is filtered through a membrane filter (0.22 .mu.m in pore 
diameter). The filtrate is distributed aseptically in 1-ml portions into 
vials, followed by lyophilization. The human IL-2 preparation for 
injection in each vial is dissolved in 1 ml of distilled water for 
injection prior to use. 
REFERENCE EXAMPLE 4 
The plasmid pILOT 135-8 obtained in Reference Example 1 was cleaved with 
the restriction enzyme HgiAI. The thus-obtained 1294 bp DNA fragment was 
treated with T4 DNA polymerase to have flat ends and was connected with 
EcoRI linker dTGCCATGAATTCATGGCA by using T4 DNA ligase. The thus-obtained 
DNA was digested with EcoRI to obtain a DNA fragment which additionally 
had translational start codon ATG and human IL-2 gene. 
This DNA fragment was inserted by using T4 DNA ligase into ptrp 781 
[Nucleic Acids Research, 11, 3077 (1983)]which had been digested at the 
EcoRI-PstI site. The thus obtained expression plasmid pTF 1 had a 
translational start codon and a human IL-2 gene downstream from the trp 
promoter (FIG. 10). 
The plasmid pTF 1 was cleaved with the restriction enzyme StuI and joined 
with the BamHI linker. This plasmid DNA was treated with the restriction 
enzymes BamHI and EcoRI, followed by insertion into pTB 281, which has the 
.lambda.PL promoter at the EcoRI-BamHI site. The expression plasmid thus 
obtained was named pTB 285 (FIG. 11). 
REFERENCE EXAMPLE 5 
Escherichia coli N4830 was transformed with the plasmid pTB285 obtained in 
Reference Example 4 in accordance with the method of Cohen et al. [vide 
supra] to obtain a transformant (Escherichia coli N4830/pTB285) carrying 
said plasmid. 
EXAMPLE 3 
E. coli N4830/pTB285 obtained in Reference Example 5 was inoculated into 50 
ml of a liquid medium (pH 7.0) containing 1% Bacto tryptone (Difco 
Laboratories, USA), 0.5% Bacto yeast extract (Difco Laboratories, USA), 
0.5% sodium chloride and 7 .mu.g/ml tetracycline and placed in a 250-ml 
erlenmeyer flask. After incubation at 35.degree. C. overnight on a swing 
rotor, the culture medium was transferred to a 5-liter jar fermenter 
containing 2.5 liters of M9 medium containing 0.5% casamino acids, 0.5% 
glucose and 7 .mu.g/ml tetracycline. Incubation was then conducted with 
aeration and stirring at 35.degree. C. for 4 hours and 42.degree. C. for 
an additional 3 hours. Cells were harvested from the thus-obtained 
2.5-liter culture broth by centrifugation, frozen at -80.degree. C. and 
stored. 
By the extraction and purification of said cells according to Example 1, 
highly purified human IL-2 protein having the same properties as that in 
Example 1 (V) was obtained from the E. coli N4830/pTB285 cells mentioned 
above. 
The following references, which are referred to for their disclosures at 
various points in this application, are incorporated herein by reference. 
Science, 193, 1007-1008 (1976) 
Immunological Reviews, 51, 257-278 (1980) 
The Journal of Immunology, 123, 2928-2929 (1979) 
Nature, 268,154-156 (1977) 
The Journal of Immunology, 130, 981-987 (1983) 
ibid., 125, 1904-1909 (1980) 
ibid., 130, 1784-1789 (1983) 
ibid., 130, 1970-1973 (1983) 
European Journal of Immunology, 10, 719-722 (1980) 
Nature, 284, 278-280 (1980) 
Nature, 302, 305-310 (1983) 
Nucleic Acids Research, 11, 4307-4323 (1983) 
Biochemical and Biophysical Research Communication, 109, 363-369 (1982) 
Cell, 8, 163-182 (1976) 
Nucleic Acids Research 9, 2251-2266 (1981) 
Methods in Enzymology, 68, 41-50 (1979) 
Proc. Natl. Acad. Sci. USA, 72, 3961-3965 (1975) 
Method in Enzymology, 68, 220-242 (1979) 
Proc. Natl. Acad. Sci. USA, 74, 560-564 (1977) 
Nucleic Acids Research, 9, 309-321 (1981) 
Proc. Natl. Acad. Sci.USA, 73, 4174-4178 (1976) 
Nature, 217, 1110-1114 (1968) 
Cell, 25, 713-719 (1981) 
Proc. Natl. Acad. Sci. USA, 69, 2110-2114 (1972) 
J. Experiments in Molecular Genetics, pages 431-433 (Cold spring Harbor 
Laboratory, New York, 1972) 
Journal of Immunology, 130, 988-992 (1983) 
The Journal of Immunology, 120, 2027-2032 (1978) 
Haemostasis, 7, 183 (1978) 
J. Molecular Biology, 96, 495-509 (1975) 
Proc. Natl. Acad. Sci. USA, 75, 5765-5769 (1978) 
Nucleic Acids Research, 9, 6103-6114 (1981) 
ibid., 7, 1513-1523 (1979) 
Methods in Enzymology, 11, 197-199 (1967) 
Analytical Biochem., 67, 438-445 (1975) 
Nucleic Acids Research, 11, 3077-3085 (1983)