Recombinant human interleukin-1.alpha.

The invention relates to the cloning of the human IL-1 gene, its engineering into suitable expression vectors, transformation of host organisms with such expression vectors and production of biologically active recombinant human IL-1 by culture of such transformed cells. Additionally, the invention relates to the isolation and use of the resulting recombinant human IL-1 polypeptides.

BACKGROUND OF THE INVENTION 
Interleukin-1 (IL-1) is a protein synthesized and secreted by activated 
macrophages. As part of the body's defense mechanism against infection and 
other forms of injury, this polypeptide hormone stimulates the 
proliferation and/or differentiation of a broad spectrum of cell types 
(including T and B lymphocytes, liver cells, bone marrow cells, connective 
tissue elements, skeletal muscle, brain cells, etc.). Through its actions 
on these diverse cell populations, IL-1 modulates immune function, fever, 
liver cell function (increased synthesis and secretion of acute phase 
reactants; increased uptake of amino acids, iron and zinc), production and 
release of neutrophils from the bone marrow, skeletal muscle proteolysis, 
changes in connective tissue, etc. IL-1 has also been described in the 
prior art as lymphocyte activating factor (LAF), leukocyte endogeneous 
mediator (LEM), endogeneous pyrogen (EP), and mononuclear cell factor 
(MCF). Until recently all studies of IL-1 were conducted with partially 
purified protein preparations, therefore, it has not been certain whether 
all the activities associated with IL-1 are contained within one molecule, 
or whether fragments of IL-1 or other macrophage proteins are responsible 
for certain of the functions outlined above. 
Since it has been difficult to prepare sufficient amounts of human IL-1 for 
structural and activity studies, the biochemical nature of this molecule 
is poorly understood. IL-1 preparations show evidence of size and charge 
heterogeneity. IL-1 activity is associated with single polypeptide chains 
with molecular weights anywhere in the range between 12,000 and 19,000. 
Recently, the gene coding for mouse IL-1 was cloned, sequenced, and 
expressed in Escherichia Coli. See in this regard Lomedico et al., Nature 
312, pp. 458-462 (Nov. 29, 1984). In conjunction with the sequencing 
studies on purified "natural" mouse IL-1, it is possible to understand how 
this hormone is synthesized to yield the population of molecules 
possessing size and charge heterogeneity. When purified natural mouse IL-1 
is electrophoresed on SDS-polyacrylamide gels, one finds multiple 
polypeptides with molecular weights between 12,000 and 19,000, all of 
which are biologically active. These polypeptides have different 
amino-terminal sequences and demonstrate charge heterogeneity on 
Tris-glycinate polyacrylamide gels. Sequencing of the cloned mouse IL-1 
cDNA and in vitro translation experiments proved that IL-1 is initially 
synthesized as a precursor polypeptide of 270 amino acids. Biologically 
active IL-1 can be obtained from E. coli by expressing the 
carboxy-terminal 156 amino acids of this precursor. Hence, IL-1 activity 
is proteolytically released from the carboxy-terminus of the 270 amino 
acid precursor protein. Multiple points of protease attack will generate a 
population of molecules with "ragged" amino-termini, thus providing an 
explanation of the size and charge heterogeneity observed in purified 
"natural" IL-1. 
The cloning of a putative gene for human IL-1 was described by Auron et 
al., Proc. Natl. Acad. Sci. USA, 81, 7907 (1984) which was published in 
February 1985. The DNA and protein sequences described therein are only 
partially homologous to the sequences described below. 
The purification of natural human IL-1 to homogeneity has been reported by 
Lachman, Fed. Proc. 42, No. 3, 2639-2645 (June 1983). The method used 
molecular weight fractionation, isoelectric focusing and preparative 
polyacrylamide gel electrophoresis. Due to the use of sodium dodecyl 
sulfate in the last step, the product was denatured and exhibited only a 
trace of its original biological activity. See also Schmidt. J. Exp. Med. 
160, 772-787 (September 1984) for a purification scheme using HPLC methods 
to produce a single charged species of human IL-1 and Kronheim et al., J. 
Exp. Med. 161, 490-502 (March 1985). 
It has also been known in the art to produce antibodies directed against 
murine IL-1. See Mizel et al. J. Immun. 131, 1834 (1983). These 
antibodies, which were raised in goat, were utilized to develop an assay 
for IL-1 and also in the production of an anti-IL-1 immunoabsorbent column 
which in turn is useful for further purification of either natural or 
recombinant murine IL-1. The anti murine IL-1 antibody crossreacts poorly 
with human IL-1. 
SUMMARY OF THE INVENTION 
The present invention relates to the cloning of the human IL-1 gene, its 
engineering into suitable expression vectors, transformation of host 
organisms with such expression vectors and production of biologically 
active recombinant human IL-1 by culture of such transformed cells. 
Additionally, the present invention relates to the isolation and use of 
the resulting recombinant human IL-1 polypeptide. 
Thus, the present invention utilizes recombinant DNA technology as the 
means to discover the DNA sequence and the deduced amino acid sequence for 
human interleukin-1 and to its production and to its use. 
More particularly, the present invention relates to the isolation and 
identification of DNA sequences coding for biologically active forms of 
human interleukin-1. This was accomplished by employing a mouse IL-1 cDNA 
clone to isolate a partial human IL-1 genomic clone. This genomic clone 
was used in turn to isolate a human IL-1 cDNA clone. The sequence of this 
cDNA revealed the structure of the human IL-1 precursor protein. 
Expression of the carboxy-terminal 154 amino acids of this precursor in E. 
coli resulted in the production of biologically active IL-1 protein. 
Thus, more particularly, the present invention relates to the isolation and 
identification of DNA sequences encoding the human IL-1 precursor and 
biologically active molecules contained therein, and to the construction 
of recombinant DNA expression vehicles containing such DNA sequences 
operatively linked to expression--effecting promoter sequences and to the 
expression vehicles so constructed. In another aspect, the present 
invention relates to host culture systems, such as various microorganism 
and vertebrate cell cultures transformed with such expression vehicles and 
thus directed in the expression of the DNA sequences referred to above. In 
other aspects, this invention relates to the means and methods of 
converting the end products of such expression to novel entities, such as 
pharmaceutical compositions, useful for the prophylactic or therapeutic 
treatment of humans or in diagnostic assay systems. In preferred 
embodiments, this invention provides particular expression vehicles that 
are constructed properly such that human interleukin-1 is produced in the 
host cell in mature form.

BRIEF DESCRIPTION OF THE PREFERRED EMBODIMENT 
As indicated above the cloned gene coding for a human IL-1 polypeptide may 
be obtained by use of a mouse IL-1 cDNA clone as a hybridization probe. In 
such a procedure, an EcoRl partial human genomic phage library (Fritsch et 
al. cell 19, 959-972 (1980)) was screened using as the hybridization probe 
the plasmid pIL-1 1301 (Lomedico et al. supra). Phage plaques were 
transferred to nitrocellulose filters by standard methods (Maniatis et al. 
Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, Cold 
Spring Harbor, N.Y., 1982) and the filters containing the imobilized DNA 
were hybridized (10.sup.6 cpm/10 ml/138 mm filter) in 5.times.SSPE 
(1.times.SSPE=0.18M NaCl, 10 mM Na phosphate pH 7.0, 1 mM Na.sub.3 EDTA), 
5.times. Denhardt's (0.1% Ficoll 400, 0.1% bovine serum albumin, 0.1% 
polyvinylpyrrolidine (w/v)), 0.3% SDS, 20% formamide, 250 microgram/ml of 
calf thymus DNA, and .sup.32 P-pIL-1 1301 (labelled by nick translation to 
1-2.times. 10.sup.8 cpm/microgram) for 48 hours at 37.degree. C. The 
filters were washed at 30.degree. C. in 0.5.times.SSPE and 
autoradiographed. After screening 7.9.times.10.sup.5 plaques, 2 positive 
recombinant phage were identified. These two phage were shown to be 
identical by restriction endonuclease mapping and named .lambda.-hil 4. 
Using the hybridization conditions described above, the mouse IL-1 cDNA 
clone pIL-1 1301 was subsequently shown to specifically hybridize to a 1.4 
kb EcoR1-Hind III fragment from the recombinant phage .lambda.-hil 4. This 
1.4 kb fragment was subcloned into pBR322 to yield phil #4. The nucleotide 
sequence proximal to the EcoR1 site of phil #4 was determined (see FIG. 
2A) and compared to the sequence of mouse IL-1 mRNA and protein. This 
analysis showed 75% nucleic acid homology and 66% amino acid sequence 
homology with the carboxy-terminal 61 amino acids of the mouse IL-1 
precursor and its accompanying 3' non-coding region. 
The partial human IL-1 gene obtained as above was then employed as a probe 
to isolate a human IL-1 cDNA clone derived from mRNA obtained from induced 
normal human peripheral blood leukocytes. The human leukocyte 
concentrates, collected and prepared from normal donors were obtained from 
the American Red Cross, Lansing, Mich. The contents from 50-100 leukocyte 
concentrates were aseptically removed from the collection bags and pooled. 
The leukocyte pool was mixed with a half volume of a 6% solution of 
hetastarch (Hespan, American Hospital Supply Corp., Irvine, Calif.) in a 
separatory funnel and allowed to stand for 3-3.5 hours at room 
temperature. This procedure differentially sedimented the contaminating 
red blood cells from the leukocytes. The volume of the leukocyte--Hespan 
mixture should not exceed two-thirds of the volume of the separatory 
funnel to insure proper sedimentation. The red blood cells sedimented to 
the bottom of the separatory funnel and cell separation was complete when 
a sharp interface band of white cells was apparent just above the red cell 
layer. The "low density" white blood cells were used for the production of 
interleukin-1. These cells were removed from the separatory funnel by 
carefully aspirating only the uppermost layer of cells located above the 
interface white cells. The low density leukocytes were removed from the 
Hespan by sedimentation in 250 ml conical centrifuge tubes (Corning 
Glassworks, Corning, N.Y.) at 500.times.g for 20 minutes. The pellet was 
resuspended in 9 volumes of a 0.83% solution of ammonium chloride for 5 
minutes to lyse the remaining red blood cells. The leukocytes were removed 
from the ammonium chloride solution by sedimentation at 500.times.g for 10 
minutes as described above. 
The cell pellet was then resuspended to a concentration not exceeding 
3.5.times.10.sup.7 cells/ml in RPMI-1640 medium (GIBCO) prewarmed to 
37.degree. C. No fetal calf serum was added to the medium to avoid 
clumping of the concentrated cells. The freshly isolated leukocytes, free 
of red blood cell contamination, were diluted in the induction vessel with 
the appropriate volume of RPMI-1640 medium supplemented with 2% fetal calf 
serum to make a final concentration of 3.times.10.sup.6 cells/ml. The 
cells were incubated at 37.degree. C. for one hour and induced for 
production of IL-1 by the addition of 10 .mu.g/ml E.coli 
lipopolysaccharide B (LPS, Difco 3880-25). Incubation was continued for 12 
hours for mRNA extraction. The cells were then removed from the induction 
medium by sedimentation at 500.times.g. Cells induced for mRNA extraction 
may be stored as a frozen pellet at minus 20.degree. C. or lower. 
A particularly preferred procedure for isolating poly(A).sup.+ RNA from 
human peripheral blood leukocytes induced with LPS is the guanidine 
thiocyanate-CsCl method of Chirgwin et al. (Biochemistry 18, 5294-5299, 
1979) and oligo (dT)-cellulose chromatography (Aviv and Leder, Proc. Natl. 
Acad. Sci. 69, 1408-1412, 1972). Northern blot analysis of this RNA 
demonstrated that clone phil #4 specifically hybridized to a mRNA 
approximately 2,100 nucleotides long. Total poly (A) RNA was fractionated 
by sucrose density gradient centrifugation and mRNA with sizes between 
1,000 and 3,000 nucleotides was collected. Using this enriched mRNA pool, 
a cDNA library of approximately 20,000 clones was constructed in pBR 322 
by established procedures (see Gubler and Hoffman, Gene 25, 263-269, 
1983). This library was screened using as a hybridization probe the 1.4 kb 
EcoR1-Hind III insert from plasmid phil #4. Bacterial colonies were 
transferred to nitrocellulose filters by standard methods (Maniatis et al. 
supra) and the filters containing the immobilized DNA were hybridized in 
5.times.SSPE, 5.times. Denhardts, 0.3% SDS, 50% formamide (w/v), 250 micro 
g/ml calf thymus DNA and P-phil #4 insert DNA for 16 hours at 37.degree. 
C. The filters were washed at 50.degree. C. in 0.1.times.SSPE and 
autoradiographed to identify a single positive clone termed phil #7. This 
clone contains an insert of 2,200 bp. The nucleotide sequence of this 
insert (see FIG. 2A) contains the partial sequence of the genomic clone 
phil #4 and hence corresponds to the cDNA sequence of mRNA encoded by this 
gene. The nucleotide sequence of this phil #7 insert predicts an open 
reading frame for a protein of 163 amino acids. This predicted protein is 
55% homologous (see FIG. 3) to the carboxy-terminal 160 amino acids of the 
mouse IL-1 precursor. Hence the sequence provided by phil #7 represents 
the carboxy-terminal region of the human IL-1 precursor. Primer extension 
experiments demonstrate that approximately 400 nucleotides from the 5' end 
of the human IL-1 mRNA are missing from clone phil #7. 
Since human IL-1 cDNA clone #7 turned out to be an incomplete copy of human 
IL-1 mRNA, the following strategy to complete the sequence was adapted: 
Based on the DNA sequence of clone #7, a synthetic oligonucleotide with 
the sequence 5'-GGGCGTCATTCAGGATGAATTCGTA-3' was devised and synthesized 
using solid support phosphoramidite technology. This oligonucleotide is 
complementary to the sequence coding for amino acids 20-28 as predicted 
from phil #7. The oligonucleotide spans a region in the cDNA predicted to 
contain an EcoRI restriction site. Such a site is useful for fusing an 
extension clone with clone #7 to create a cDNA encompassing the complete 
protein coding region for the human IL-1 precursor. This oligonucleotide 
was annealed to size-fractionated poly A.sup.+ RNA from LPS--induced 
human peripheral blood leukocytes (supra). Annealing conditions were 50 mM 
NaCl, 10 mM DTT, 0.05 mM EDTA, 550 pmoles oligonucleotide/ml, 250 mcg of 
poly A.sup.+ RNA/ml for one minute at 90.degree. C., 10' at 43.degree. C., 
10' at 20.degree. C. after which the reaction was cooled on ice. 
cDNA--synthesis and establishment of an extension cDNA--library were 
performed as described above for clone #7. About 10.sup.5 independent 
transformants were generated in this way from 5mcg of poly A.sup.+ RNA 
enriched for human IL-1 mRNA. A total of approximately 2900 were screened 
with the oligonucleotide described above that had been labelled with 
polynucleotide kinase and .gamma.-.sup.32 P-ATP according to standard 
procedures (Maniatis et al., supra). Colony bearing nitrocellulose filters 
were made according to standard procedures (Maniatis et al., supra). The 
filters were hybridized with the labelled oligonucleotide under the 
following conditions: 5.times.SSPE, 10.times. Denhardts, 0.1% SDS, 100 
.mu.g/ml yeast soluble RNA; 0.2 pmoles/ml of labelled oligonucleotide 
(specific activity=.about.1 .mu.Ci/pmole) for 15' at 65.degree. C. and 
subsequently 2 hours at 37.degree. C. The filters were then washed twice 
in 2.times.SSPE-0.025% SDS (quick rinses at room temperature) and then in 
4.times.SSPE-0.025% SDS at 51.degree. C. for 60'. The filters were then 
air dried, exposed to x-ray film with the aid of an intensifying screen at 
-70.degree. C. for 16 hours. Twelve positive colonies were further 
analyzed by restriction endonuclease cleavage. Clone phil #19 was chosen 
for further analysis. The sequence of the insert (see FIG. 2B) from phil 
#19 contains the expected overlap with phil #7 and predicts a single open 
reading frame coding for 139 amino acids. This region represents the 
amino-terminal end of the human IL-1 precursor protein, and is highly 
homologous to the corresponding region of the mouse IL-1 precursor protein 
(FIG. 3). Hence, combining the sequence information from phil #7 and phil 
#19. human IL-1 mRNA codes for a protein of 271 amino acids which is 
significantly related to the 270 amino acid mouse protein (FIG. 3). 
Plasmid phil #7 contains the coding information for the carboxy-terminal 
163 amino acids of the 271 amino-acid human IL-1 precursor protein. 
Plasmid phil #19 contains the coding information for the amino-terminal 
139 amino acids of this protein. Each plasmid possesses a single EcoR1 
restriction endonuclease cleavage site within the sequence (94 nucleotides 
long) that is common to their inserts. This EcoR1 site can be used to join 
the information from the two plasmids into a single composite plasmid 
containing the entire coding region from the human IL-1 precursor protein. 
Using standard methods, plasmids phil #7 and phil #19 can be individually 
digested with EcoR1 and Bam HI, and the resultant DNA fragments separated 
by polyacrylamide gel electrophoresis. The .about.2100 bp EcoR1-Bam HI 
fragment can be isolated from the phil #7 digest, and the .about.460 bp 
Bam HI--EcoR1 fragment can be isolated from the phil #19 digest. The two 
isolated fragments can be ligated together using T4 DNA ligase. The ligase 
is heat-inactivated and the mixture is treated with Bam HI. This mixture 
can be ligated to Bam HI-linearized pEV-vrf2 (below) and used to transform 
E.coli strain MC1061 containing the compatible plasmid pRK248 (cIts) using 
selection for ampicillin resistance. Bacterial clones can be screened by 
restriction endonuclease cleavage analysis to identify a plasmid, phil 
#1-271*, which contains the expected insert in the correct orientation. 
Plasmid phil #1-271* is modified by site-directed oligonucleotide 
mutagenesis (see below) to remove the extraneous nucleotides between the 
initiation ATG codon and the alanine codon (GCC) which is the second amino 
acid in the 271 amino acid precursor protein, to generate phil #1-271. 
Bacteria containing phil #1-271, when induced by temperature shift 
(supra), synthesize the complete 271 amino acid IL-1 precursor protein as 
set forth in FIG. 2B. 
Plasmid pEV-vrf2 is a pBR322 derivative modified using synthetic DNA 
oligonucleotides to contain a ribosome binding site--initiation codon 
downstream from a tightly regulated phage .lambda. P.sub.L promoter. 
Multiple-use restriction endonuclease cleavage sites exist immediately 
downstream from the initiation codon, allowing for the insertion of coding 
region sequences to be expressed as fusion proteins with 2-9 extra 
amino-terminal amino acids. These extraneous amino acids can be removed by 
site directed mutagenesis, resulting in the expression of the desired 
protein. The genealogy of pEV-vrf2 is as follows: 
pBR322.fwdarw.pRC2.fwdarw.pRC23.fwdarw.pEV-vrf2. 
pRC2 is a derivative of pBR322 containing a unique BglII site adjacent (on 
the amp.sup.R side) to the EcoRI site in the plasmid. This plasmid was 
constructed using known methods in the following manner. 20 .mu.g of 
pBR322 plasmid DNA was digested with EcoRI and then split into two 
reactions. In one, the protruding 5' single-stranded termini were removed 
with S1 nuclease; in the other reaction, the termini were filled-in by 
incorporating deoxynucleotides with the Klenow fragment of DNA polymerase 
I. Both reactions were terminated by phenol extraction followed by ethanol 
precipitation. Approximately 1 .mu.g of DNA from each reaction was mixed 
with 90 pmoles of phosphorylated BglII linkers (CAGATCTG, purchased from 
Collaborative Research) and incubated with T4 DNA ligase at 15.degree. for 
18 hours. The ligation products were then digested with BglII and PstI and 
subjected to gel electrophoresis in 1% agarose. The 3600 bp and 760 bp 
fragments from both reactions were recovered from the gel. For the 
construction of pRC2, the 3600 bp from the Klenow reaction was ligated to 
the 760 bp fragment from the S1 reaction. E.coli strain RR1 was 
transformed with the ligation mixtures, and transformants were selected on 
LB agar plates containing 50 .mu.g/ml ampicillin. Transformants containing 
the expected plasmid constructions were identified by restriction analysis 
of the isolated plasmid DNA. DNA sequence analysis confirmed that the S1 
nuclease treatment precisely removed the 5' single-stranded termini. 
pRC23 was constructed by inserting into pRC2 a 250 bp BglII-HaeIII fragment 
containing the .lambda. P.sub.L promoter joined to a pair of complementary 
synthetic oligonucleotides comprising a model ribosome-binding site (RBS). 
The HaeIII site is located within the 5' non-coding region of the 
.lambda.N gene 115 bp downstream of the P.sub.L transcription initiation 
site (Sanger, et al., 1982). Approximately lg of a 450 bp BglII-HpaI 
fragment isolated from phage .lambda. DNA was digested with HaeIII. 200 ng 
of the resulting digestion products were mixed with 60 pmoles each of 
phosphorylated synthetic oligonucleotides containing the model RBS. These 
complementary deoxynucleotides (#1=TTAAAAATTAAGGAGG; 
#2=AATTCCTCCTTAATTTTTAA) were synthesized on solid support using the 
phosphite methodology (Matteucci, M. D. and Caruthers, M. H. "Synthesis of 
Deoxyoligonucleotides on a Polymer Support" J. Am. Chem. Soc. (1983) 
103:3185-3191). Synthesis was initiated with 1 .mu.mole of the 3'-terminal 
nucleoside attached to a controlled pore glass support (Pierce CPG/long 
chain alkylamine resin). This mixture was incubated with T4 DNA ligase at 
15.degree. C. for 18 hours, and the ligated molecules were digested with 
BglII and EcoR1 and separated on a 5% polyacrylamide gel. The 270 bp 
ligation product was recovered from the gel, mixed with gel purified pRC2 
vector that had been digested with BglII and EcoRI, and incubated with T4 
DNA ligase at 15.degree. for 15 hours. The ligation mixture was used to 
transform strain RR1 (pRK248cIts). Transformants selected on 
ampicillin-containing medium were screened by restriction analysis of the 
isolated plasmid DNA. The expected plasmid construction, pRC23, was 
confirmed by further restriction enzyme digestions and by DNA sequence 
analysis across the EcoRI junction. Plasmid pRC23 contains a unique EcoRI 
site at the 3' end of the RBS, into which genes containing an ATG at the 
5' end can be inserted. 
For the construction of pEV-vrf2, pRC23was digested with EcoRI and Hind III 
and the linearized vector isolated by preparative agarose gel 
electrophoresis. Two complementary deoxynucleotides 
(#3=AATTAATATGAATAGAATTCGGATCCATCGATA, 
#4=AGCTTATCGATGGATCCGAATTCTATTCATATT) were synthesized (supra) combined 
and heated to 58.degree. C. for 5 minutes in 150 mM NaCl, and cooled 
slowly to allow annealing. 0.1 pmoles of the synthetic duplexes were added 
to 0.07 pmoles of the pRC23/EcoRI-HindIII vector and incubated with T4 DNA 
ligase at 15.degree. C. for 15 hours. Strain RR1 (pRK248cIts) was 
transformed with the ligation products, and ampicillin-resistant 
transformants were screened by restriction endonuclease cleavage analysis 
to identify pEV-vrf2, the expected construction of which was confirmed by 
DNA sequence analysis. Plasmid pEV-vrf2 contains restriction sites (EcoRI, 
BamHI, ClaI, and HindIII) located downstream from an appropriately 
positioned initiation codon--RBS. Hence, appropriately positioned coding 
region sequences inserted into these restriction sites will be expressed 
under control of the P.sub.L promoter yielding the corresponding protein 
with 2-9 extra amino-terminal amino acids. Site directed mutagenesis can 
be used to remove these extraneous amino acids, as well as to re-orient 
inappropriately positioned (i.e. the reading frame is not correct) coding 
sequences. 
Synthetic oligonucleotides 5'-GGGCGTTATTCAGGACGAATTCGTA-3' and 5'-ATTGCTCA 
GG AACATATTAATCC-3' used above were synthesized on an Applied Biosystems 
model 380A DNA synthesizer. Diisopropyl phosphoramidites were used as the 
active nucleotidyl component for coupling to the support. See Beacage and 
Caruthers, Tetrahedron Lett. 22, 1859-1862 (1981). Synthesis on the 
support was carried out at 0.5 mmol level using a CPG resin with the 3' 
nucleotide attached to the resin as described by Matteucci and Caruthers, 
J. Am. Chem. Soc. 103, 3185-3189 (1981). The synthetic cycle was 
essentially the same as provided by the manufacturer, however, 2% 
dichloroacetic acid was used in place of 3% trichloroacetic acid as the 
detritylating reagent. Synthesized oligonucleotides were isolated on 20% 
polyacrylamide sequencing gels. Isolated oligonucleotides cut out from the 
gel were eluted in gel elution buffer and desalted on C.sub.18 
reverse-phase columns. 
With the recombinant DNA thus obtained, living cells may be transformed to 
amplify the cloned cDNA or to produce IL-1 polypeptide. 
Suitable eucaryotic host organisms, which may be employed for production of 
IL-1, include vertebra, yeast and the like. For instance, monkey cells, 
e.g. CV-1 cells, transformed by a replication origin defective mutant of 
SV-40 and expressing the SV-40 large T antigen (COS cells) as discussed by 
Gluzman (Cell 23, 175-182, 1981), mouse derived cells described by Ohno 
and Taniguchi (Nucleic Acids Research 10, 967-977 (1982)), and yeast 
host--vector systems which have been utilized for the expression of 
interferon genes, discussed by Hitzman et al. (Nature, 293, 717-722 
(1981)) may be utilized. In addition, it is possible to use insect cells 
such as described by Smith et al. (Mol. Cell. Biol. 3, 2156-2165, 1983). 
Suitable procaryotic host organisms include Escherichia coli, Bacillus 
subtilis and the like. For amplification of DNA in host organisms, it may 
be preferred to use E. coli as a host, however other hosts can also be 
employed. 
Suitable vectors used for E. coli include EK type plasmid vectors 
(stringent type): pSC101, pRK353, pRK646, pRK248, pDF41 etc., EK type 
plasmid vectors (relaxed type): ColEI, pVH51, pAC105, RSF2124, pCR1, pMB9, 
pBR313, pBR322, pBR324, pBR325, pBR327, pBR328, pKY2289, pKY2700, pKN80, 
pKC7, pKB158, pMK2004, pACYCl, pACYC184, dul etc. .lambda. gt type phage 
vectors: .lambda. gt. .lambda.c, .lambda. gt, .lambda.B, .lambda. WES, 
.lambda.c, .lambda. gt. .lambda.B, .lambda.WES, .lambda.C, .lambda.WES, 
.lambda.B, .lambda.ZJvir., .lambda.B', .lambda.ALO, .lambda.B, 
.lambda.WES. Ts622, .lambda.Dam etc. In general pBR322 has been frequently 
used as a vector for E. coli. 
Transformation of the host cell with the recombinant DNA may be carried out 
by conventionally used methods as follows: 
Where the host is prokaryotic such as E. coli, competent cells which are 
capable of DNA uptake are prepared from cells harvested during the 
exponential growth phase and subsequently treated by the CaCl.sub.2 
-method by well known procedures. When MgCl.sub.2 or RbCl exists in the 
transformation reaction medium, the transformation efficiency increases. 
Transformation can be also performed after forming a protoplast of the 
host cell. 
Where the host used is eucaryotic, transfection methods of DNA as calcium 
phosphate-precipitates, conventional mechanical procedures such as 
microinjection, insertion of a plasmid encapsulated in red blood cell 
hosts or in liposomes, treatment of cells with agents such as 
lysophosphatidylcholine, or use of virus vectors, or the like may be used. 
However, various other microbial strains are useful, including known E. 
coli strains such as E. coli B. E. coli X 1776 (ATCC No. 31537) and E. 
coli W 3310 (ATCC No. 27325), and most preferably E. coli RR1' or other 
microbial strains such as MC 1061, many of which are deposited and 
available from depository institutions, such as the American Type Culture 
Collection (ATCC)-cf. the ATCC catalogue listing. See also German 
Offenlegungsschrift 2644432. These other microorganisms include, for 
example, Bacilli such as Bacillus subtilis and enterobacteriaceae among 
which can be mentioned as examples Salmonella typhimurium and Serratia 
marescens, utilizing plasmids that can replicate and express heterologous 
gene sequences therein. 
As examples, the beta lactamase and lactose promoter systems have been 
advantageously used to initiate and sustain microbial production of 
heterologous polypeptides. Details relating to the make-up and 
construction of these promoter systems have been published by Chang et 
al., Nature 275, 617 (1978) and Itakura et al., Science 198, 1056 (1977), 
which are hereby incorporated by reference. More recently, a system based 
upon tryptophan, the so-called trp promoter system, has been developed. 
Details relating to the make-up and construction of this system have been 
published by Goeddel et al., Nucleic Acids Research 8, 4057 (1980). 
Numerous other microbial promoters have been discovered and utilized and 
details concerning their nucleotide sequences, enabling a skilled worker 
to ligate them functionally within plasmid vectors, have been 
published-see, e.g., Siebenlist et al., Cell 20, 269 (1980). 
The expression system hereof may also employ the plasmid YRp7, which is 
capable of selection and replication in both E. coli and the yeast, 
Saccharomyces cerevisiae. A useful strain is RH218 deposited at the 
American Type Culture Collection without restriction (ATCC No. 44076). 
However, it will be understood that any Saccharomyces cerevisiae strain 
containing a mutation which makes the cell trp1 should be an effective 
environment for expression of the plasmid containing the expression 
system. An example of another strain which could be used is pep4-1. This 
tryptophan auxotroph strain also has a point mutation in the TRP1 gene. 
The experience with expression of mouse IL-1 cDNA in E. coli suggests that 
the carboxy-terminal portion of the human IL-1 precursor should possess 
IL-1 biological activity. Clone phil #7 described above, contains the 
coding information for the carboxy-terminal 163 amino acids of the human 
IL-1 precursor. There is an Alu I restriction endonuclease cleavage site 
near the 5' end of the phil #7 insert (see FIG. 2A) within the codon for 
the ninth amino acid (that is, the 153 amino acid from the 
carboxy-terminal end of the precursor). The next downstream Alu I site is 
in the 3' non-coding region (that is, past the termination codon) 
.about.600 bp away. This .about.600 bp Alu I fragment containing the 
sequences coding for the carboxy-terminal 154 amino acids of the human 
IL-1 precursor, was isolated from phil #7 and inserted into the BamHI site 
of an E. coli expression plasmid (as seen in FIG. 4) in the following 
manner. Using standard methods, the insert from clone phil #7 was digested 
with Alu I, and phosphorylated BamHI linkers (CGGATCCG New England 
Biolabs, Catalog 1021) were ligated to the Alu I cut insert using T4DNA 
ligase. The ligase was heat-inactivated and the mixture was treated with 
BamHI to remove excess linkers and to generate cohesive termini. This 
mixture was electrophoresed on a polyacrylamide gel and the .about.600 bp 
fragment was isolated. Plasmid pEV-vrf2 was digested with BamHI and the 
linearized vector was recovered following agarose gel electrophoresis. The 
.about.600 bp fragment and the BamHI cut vector were ligated together, and 
used to transform E. coli strain MC 1061 (Casadaban and Cohen J. Mol. 
Biol. 138, 179-207, 1980) containing the compatible plasmid pRK248cIts 
(Bernard and Helinski, Meth. Enzym. 68, 482-492, 1979) using selection for 
ampicillin resistance. Bacterial clones were screened by restriction 
endonuclease digestion analysis to identify a plasmid phil #1-154* 
containing the insert in the correct orientation. Plasmid phil #1-154* was 
partially sequenced to verify that its structure was correct. Bacteria 
containing phil #1-154* or the parental plasmid pEV-vrf2 were grown in M9 
media containing ampicillin at 30.degree. C. until the A.sub.550 reached 
0.7, at which time the cultures were shifted to 42.degree. C. for 3 hours. 
The bacteria from 1 ml of culture were recovered by centrifugation and 
solubilized in 50 micro liters of 7M guanidine hydrochloride. These crude 
bacterial extracts were examined for IL-1 activity in the murine thymocyte 
proliferation assay (Mizel et al. supra). Extracts of bacteria containing 
only pEV-vrf2 did not stimulate in the assay above background levels. 
Extracts of bacteria 10 containing phil #1-154* contained 32,000 units of 
IL-1 activity/ml of guanidine HCl solution. Assuming a specific activity 
of 6.times.10.sup.6 units per mg, this is equivalent to at least 0.3 mg of 
IL-1 protein per liter of bacterial culture. 
The protein encoded by expression plasmid phil #1-154* contains, in 
addition to the initiator methionine, 6 extraneous amino acids at its 
amino terminus. These were removed by site directed mutagenisis as 
follows: 
1-2 .mu.g of plasmid phil#1-154* was subjected to restriction endonuclease 
digestion in two separate reactions. In one reaction, 1-2 units of both 
Bgl II and Bam HI created a linearized plasmid with a gap (see FIG. 5), 
and in the other reaction, 1 unit of Pst I generated a linearized plasmid. 
Pst I treatment was followed by treatment with 1 unit of Klenow fragment 
of E.Coli DNA polymerase I. These opened plasmids were purified by 
electrophoresis through a 0.7% aparose gel and recovered by ethanol 
precipitation. Each plasmid was resuspended in 5 .mu.l H.sub.2 O. A 1 
.mu.l aliquot was taken from each and was combined with 50 ng of 
phosphorylated synthetic oligonucleotide: 
5'-P-ATTGCTCAGGAACATATTATTCC-OH-3' in a 12 .mu.l reaction containing 12 mM 
Tris.HCl, pH 7.5, 9 mM MgCl.sub.2. 200 mM NaCl and 20 .mu.l beta 
mercaptoethanol. The reaction was heated to 100.degree. C. for 3' to 
denature the opened plasmids, and the annealing of the oligonucleotide was 
permitted by gradually cooling the reaction at 23.degree. C. for 30', 
followed by 4.degree. C. for 30' and 0.degree. C. for 10'. This resulted 
in the formation of a heteroduplex form of phil #1-154* with the 
oligonucleotide annealed to the single-stranded region. 
The single-stranded region was made double-stranded and the plasmid was 
ligated in a 20 .mu.l reaction volume of 75 .mu.M dATP, 75 .mu.M dTTP, 75 
.mu.M dCTP, 75 .mu.M dGTP, 500 .mu.M ATP, 2-3 units of the Klenow fragment 
of E.Coli DNA polymerase I and 1 unit of T.sub.4 DNA ligase. This reaction 
proceeded at 15.degree. C. for 12-16 hours. Plasmid DNA was recovered by 
ethanol precipitation and resuspended in 10 .mu.l H.sub.2 O. A 5 .mu.l 
aliquot was used to transform the MC1061 strain of E.Coli containing the 
compatible plasmid pRk248cIts to ampicillin resistance. Ampicillin 
resistant transformants were screened for the new plasmid phil #1-154 in 
the phil #1-154* background by recovering plasmid DNA from individual 
transformants and performing Bgl II/Bam HI restriction digestion on this 
plasmid DNA preparation. Plasmid DNA which contained phil #1-154was used 
in a second round of MC 1061 pRK248cIts transformation to separate phil 
#1-154and phil #1-154*. Transformants containing only phil #1-154were 
recovered and an individual E. Coli colony was used for the production of 
the phil #1-154protein as described above. 
Purification of Recombinant Human Interleukin-1 
Like many other recombinant proteins (see Williams, D. C. et al. Science 
(1982) 215:687-688: Lacal, J. C. et al. Proc. Natl. Acad, Sci. (1984) 
81:5305-5309), human interleukin-1 aggregates into insoluble cytoplasmic 
"inclusion bodies" within E.coli. Hence the purification of recombinant 
human IL-1 begins with the isolation of these "inclusion bodies" (see 
Lacal et al., supra). 
E. coli cell paste (1 g) was suspended in 5 ml of 1 mM phenylmethylsulfonyl 
fluoride in buffer A (30 mM Tris-HCl, pH 8, in 5 mM EDTA) and the cells 
were sonicated six times for a total of 3 minutes using a Sonifier cell 
disrupter Model 350 (Branson Sonic Power Co.). The cell lysate was 
centrifuged for 30 minutes at 30,000.times.g to separate the insoluble 
fraction. The particulate fraction (which contains most of IL-1 activity) 
was sequentially washed with 5 ml each of 1) buffer A, 2) 1% Triton X-100 
in buffer A and 3) 1.75M guanidine HCl. After each wash, the particulate 
fraction was pelleted by centrifugation at 30,000.times.g for 20 minutes. 
IL-1 activity was solubilized from the remaining particulate fraction by 3 
ml of 5M guanidine HCl, followed by centriguation at 30,000.times.g for 30 
minutes. Up to this step, all procedures were carried out at 4.degree. C. 
The solubilized IL-1 protein was purified to homogeneity by gel filtration 
chromatography on Sephacryl S-200 or Sephadex G-75 (Pharmacia Fine 
Chemicals, Piscataway, N.J.) equilibrated and eluted with 5M guanidine 
HCl. The purified 1-154* IL-1 behaved as a single polypeptide on SDS 
polyacrylamide gels (Laemmli, U.K. Nature (1970) 227:680-685). When 
submitted for amino acid compositional analysis and amino-terminal 
sequence analysis, the expected results were obtained, thus verifying the 
purity and identity of the protein. In similar fashion E.coli transformed 
with phil #1-154were used to produce 1-154 protein which was purified as 
above. 
Experiments with the expression products of deletion mutants of the mouse 
IL-1 gene has provided a basis for determining the minimum sequence which 
provides a bioactive protein from the carboxy-terminal of the mouse IL-1 
precursor. The results are summarized below in Table I. 
TABLE I 
______________________________________ 
Activity of Mouse IL-1 
Deletion Mutants 
Protein.sup.1 
Activity.sup.2 
______________________________________ 
1-156 6 .times. 10.sup.6 
17-156 6 .times. 10.sup.6 
30-156 0 
1-143, 156 
0 
17-143, 156 
0 
30-143, 156 
0 
______________________________________ 
1: in this nomenclature, protein 1-156 contains the carboxyterminal 156 
amino acids of the mouse IL1 precursor. All the deletion mutants are 
defined relative to this molecule, hence protein 17-156 is missing the 
aminoterminal 16 amino acids compared to protein 1-156; protein 1-143,156 
is missing amino acids 144-155 compared to protein 1-156. 
2: in the thymocyte proliferation assay, units per mg protein. 
As seen in Table I the sequence proximal to the carboxy-terminus is needed 
for activity. A minimum of about 139 amino acids is apparently required to 
maintain activity as the 17-156 exhibits high activity, whereas deletion 
of an additional thirteen amino acids at the amino-terminus of this 
fragment destroys the activity. Thus, by analogy to the mouse molecule 
data it is believed that the sequence encompassing the carboxy-terminal 
139 amino acids of the human IL-1 precursor is the minimum fragment 
exhibiting IL-1 activity. This would correspond to positions 132-271 of 
the human IL-1 precursor protein sequence set forth in FIG. 2B. Therefore, 
one aspect of the present invention relates to peptides exhibiting human 
IL-1 activity which encompass the aforesaid minimum carboxy-terminal 
sequence. 
The purified recombinant human IL-1 peptides encompassing the sequence 
needed for biological activity can be employed in a manner known per se to 
stimulate the immune system of a host subject, such as, for example, by 
improving host defense response to pathogens, by acting as a vaccine 
adjuvant and by enhancing host defense against neoplastic diseases. Other 
clinical uses identified for human IL-1 in the art include promotion of 
wound healing via stimulation of fibroblast proliferation and improvement 
of the recovery is of critically ill, protein-malnourished patients. 
Purified IL-1 peptides prepared in accordance with this invention may be 
administered to warm blooded mammals for the clinical uses indicated 
above. The administration may be by any conventional method such as by 
parenteral application either intravenously, subcutaneously or 
intramuscularly. Obviously, the required dosage will vary with the 
particular condition being treated, the severity of the condition, the 
duration of the treatment and the method for administration. A suitable 
dosage form for pharmaceutical use may be obtained from sterile filtered, 
lyophilized IL-1 peptide reconstituted prior to use in a conventional 
manner. It is also within the skill of the art to introduce buffers, 
stabilizers, bacteriostats and other excipients and additives 
conventionally employed in pharmaceutical parenteral dosage forms.