Purification of recombinant interleukin-1

A process is provided for purifying recombinant IL-1 from microbial cells, comprising suspending the cells in an aqueous buffered medium having a pH from about 1 to about 5; disrupting the cells to provide an extract containing solubilized IL-1; and recovering solubilized Il-1 from the extract.

BACKGROUND OF THE INVENTION 
The present invention relates generally to protein chemistry, and 
specifically to processes for purifying reoombinant proteins produced by 
high level expression in microorganisms. 
Interleukin-1 (IL-1) is a lymphokine released by macrophages in response to 
immunogenic stimulation. This polypeptide has been associated with a 
complex spectrum of biological activities. IL-1 is a primary 
immunostimulatory signal capable of inducing thymocyte proliferation via 
induction of interleukin-2 release, and stimulating proliferation and 
maturation of B-lymphocytes. In addition, IL-1 has been linked with 
prostaglandin production and induction of fever, and with promotion of 
wound healing. Reviews of the literature relating to IL-1 include 
Oppenheim et al., Immunol. Today 7:45 (1986), and Durum et al., Ann. Rev. 
Immunol. 3:263 (1985). 
Human IL-1 activity resides in two distantly related proteins, herein 
designated IL-1.alpha. and IL-1.beta.(March et al., Nature 315:641 
(1985)). Both molecules are normally synthesized as larger precursors 
having molecular weights of about 30,000 daltons, which are subsequently 
proteolytically processed to yield mature forms having molecular weights 
of approximately 17,500 daltons. 
Recently, cDNAs coding for both human IL-1 species have been cloned and 
expressed in microorganisms. This achievement should enable production of 
sufficient quantities of IL-1.alpha. and IL-1.beta. to permit therapeutic 
use. However, difficulties have been encountered in purification of 
active, nondenatured IL-1 species from cells capable of high level 
expression of the recombinant proteins. These difficulties have been 
attributed to the observation that the recombinant protein appears to be 
concentrated by the producing organisms in an insoluble form. 
Gubler et al., J. Immun. 36:2492 (1986) reported a process for crude 
purification of IL-1.alpha. expressed in E. coli. However, this process 
involved use of the denaturing agents guanidine hydrochloride and urea to 
enhance solubilization of the recombinant proteins. 
The present invention provides a rapid, efficient acid-mediated recombinant 
protein solubilization process for isolating recombinant IL-1 from 
microbial cell cultures. 
SUMMARY OF THE INVENTION 
The present invention provides a process for purifying recombinant IL-1 
from microbial cells, comprising: 
(a) suspending the cells in an aqueous buffered medium having a pH from 
about 1 to about 5; 
(b) disrupting the cells to provide an extract containing solubilized IL-1; 
and 
(c) recovering solubilized IL-1 from the extract. In a product aspect, the 
present invention provides purified recombinant IL-1.alpha. having a 
specific activity greater than about 6.0.times.10.sup.8 units per mg and 
endotoxin levels less than 100 pg endotoxin/.mu.g rIL-1.alpha.; and 
purified recombinant IL-1.beta. having a specific activity greater than 
about 1.9.times.10.sup.8 units per mg and endotoxin levels less than about 
50 pg endotoxin/.mu.g rIL-1.beta., where endotoxin levels are measured by 
a limulus amebocyte lysate assay. 
DETAILS OF THE INVENTION 
The process of the present invention involves extraction of recombinant 
human IL-1 species from microbial cells under acid conditions. Acid 
extraction simultaneously solubilizes the IL-1 and precipitates the bulk 
of the microbial proteins, enabling recovery of IL-1 in supernatants of 
the acid extracts. Preliminary experiments have indicated that in E. coli, 
recombinant IL-1 is expressed in an insoluble form. In general, 
recombinant proteins can be solubilized with such chaotropic agents as 
guanidine or urea. In contrast, acid-mediated extraction avoids denaturing 
extractants, and allows purification to proceed directly from the initial 
extraction step to subsequent ion exchange procedures without solvent 
interference or protein denaturation. This process is thus ideally suited 
for scaling-up to provide commercially significant quantities of 
recombinant IL-1.alpha. and IL-1.beta.. 
As used herein, "interleukin-1", "recombinant interleukin-1", "IL-1" and 
"rIL-1" refer collectively to recombinant forms of IL-1.alpha. and 
IL-1.beta. produced by microbial fermentation processes. In addition, the 
term comprehends proteins having amino acid sequences substantially 
identical to that of native mammalian forms of IL-1.alpha. and IL-1.beta., 
which possess biological activity in common with the native forms. 
Substantial identity of amino acid sequences means that the sequences are 
identical or differ by one or more amino acid alterations (deletions, 
additions, or substitutions) that do not cause an adverse functional 
dissimilarity between the synthetic protein and the native form. 
Preferably, the acid-mediated extraction step for rIL-1.alpha. is conducted 
in an aqueous buffered medium having a pH from about 2.0 to about 3.5, and 
most preferably, at a pH from about 2.6 to about 3.0. In the case of 
rIL-1.beta., the acid extraction step is preferably conducted at a pH from 
about 3.5 to about 4.5, and most preferably, at a pH from about 3.7 to 
about 4.1. As used herein, the term "microbial cells" means bacteria, 
particularly Escherichia coli, and yeast, e.g., Saccharomyces cerevisiae. 
In the process of this invention, cells can be disrupted by any convenient 
method, including freeze-thaw cycling, sonication, mechanical disruption, 
or use of cell lysing agents. 
In a more complete process aspect, the acid extraction steps are coupled 
with subsequent chromatography in aqueous media. This part of the 
purification process preferably includes an initial ion exchange 
chromatography stage followed by affinity chromatography. The ion exchange 
stage comprises, in a preferred aspect, cation exchange chromatography 
followed by anion exchange chromatography. 
Suitable cation exchange chromatography media include various insoluble 
matrices comprising sulfopropyl or carboxymethyl groups. Sulfopropyl 
groups are preferred. The matrices can be acrylamide, agarose, dextran, 
cellulose or other ion exchange resins or substrates commonly employed in 
protein purification. A particularly useful material for cation exchange 
chromatography of rIL-1.alpha. and rIL-1.beta. is Sulphopropyl Sephadex 
C-25 (Pharmacia Fine Chemicals, Uppsala, Sweden). When media containing 
sulfopropyl groups are employed, extracts containing rIL-1 species are 
applied at a pH of about 4.0, in a suitable buffer such as sodium citrate. 
rIL-1 species are bound by the ion exchanger, and can be eluted in more 
highly purified form by application of a weakly basic eluant, for example, 
10 mM Tris-HCl, pH 8.1. 
Suitable anion exchange chromatography media include various insoluble 
matrices comprising diethylaminoethyl(DEAE) or diethyl-(2-hydroxypropyl) 
aminoethyl (QAE) groups. DEAE groups are preferred. The matrices can be 
acrylamide, agarose, dextran, cellulose or other types commonly employed 
in protein purification. A particularly useful material for anion exchange 
chromatography of rIL-1.alpha. and rIL-1.beta. is DEAE-Sephacel 
(Pharmacia). When media containing DEAE groups are employed, extracts 
containing rIL-1 species are applied at a weakly basic pH. For example, 
the pooled rIL-1-containing fractions resulting from the previous cation 
exchange chromatography step (at a pH of about 8.1) can be applied 
directly in a suitable buffer such as Tris-HCl. rIL-1 species are bound by 
the anion exchange media, and can be eluted in more highly purified form 
by application of a salt gradient in the same buffer. It has been 
determined that rIL-1.alpha. elutes from DEAE-Sephacel at 0.17-0.22 M 
NaCl, and rIL-1.beta. at 0.075-0.155 M NaCl. Thus, gradients ranging from 
0 to 600 mM NaCl and 0 to 400 mM NaCl are useful in purifying rIL-1.alpha. 
and rIL-1.beta., respectively. 
In its most preferred aspects, the invention provides a process including 
the foregoing extraction and ion-exchange chromatography procedures 
followed by affinity chromatography. For IL-1.alpha., affinity media 
comprising pendant phenyl glycidyl ether groups (or other groups providing 
a pendant phenyl nucleus attached by an ether or amide bridge to a 
suitable substrate) are preferred. Such media include, for example, Phenyl 
Sepharose CL-4B (Pharmacia) although other insoluble matrices, for example 
dextran or cellulose derivatives, could be employed. rIL-1.alpha. is 
applied to such media in a solution containing about 0.5 to 0.7 M, and 
preferably, about 0.6 M ammonium sulfate, in a suitable buffer at a pH of 
about 8.1, and then eluted with a decreasing linear gradient of ammonium 
sulfate followed by buffer containing no salt. rIL-1.alpha. elutes from 
Phenyl Sepharose CL-4B at about 0.25-0.10 M ammonium sulfate. 
Thus, for IL-1.alpha., the most preferred process comprises suspending 
microbial cells having associated recombinant IL-1.alpha. in an aqueous 
buffered medium having a pH from about 2.0 to about 3.5; disrupting the 
cells to extract solubilized IL-1.alpha.; applying the solubilized 
IL-1.alpha. to cation exchange media at a pH from about 2.5 to about 5.0; 
eluting the IL-1.alpha. from the cation exchange media at a pH from about 
7.5 to about 9.0; applying the IL-1.alpha. to anion exchange media in a 
buffer having low osmolarity; eluting the IL-1.alpha. in a gradient of 
increasing salt concentration; applying the IL-1.alpha., in a buffer 
containing 0.5 to 0.7 M ammonium sulfate, to media comprising pendant 
phenyl glycidyl ether groups, and eluting the IL-1.alpha. in a gradient of 
decreasing ammonium sulfate concentration. The resulting fractions can be 
concentrated by a final chromatography step on media containing pendant 
sulfopropyl groups. 
For the final affinity step for purifying IL-1.beta., affinity media 
comprising pendant triazinyl red dye ligand groups are preferred. Such 
media include those having pendant groups 
##STR1## 
formed by conjugation of certain sulfonated naptholine dye species known 
as Procion Red or "reactive red", with appropriate insoluble substrates. 
Commercially available materials meeting these criteria include Red 
Sepharose CL-6B (Pharmacia), reactive red-agarose (Sigma Chemical Company, 
St. Louis, Mo., U.S.A.) and Procion Red agarose (Bethesda Research 
Laboratories, Gaithersburg, Md. U.S.A.) Preferably, pooled fractions 
containing rIL-1.beta. are applied to the dye-ligand media at a low ionic 
strength, e.g., less than 40 mM, in a suitable buffer such as 10 mM 
Tris-HCl. The media comprising bound rIL-1.beta. is then washed with 
additional application buffer, and the desired protein eluted in a linear 
gradient of increasing salt concentration, e.g., 0 to 1 M NaCl. 
rIL-1.beta. elutes from Procion Red at about 0.36-0.46 M NaCl. The 
resulting fractions can be concentrated by a final chromatography step on 
media containing pendant sulfopropyl groups. 
Thus, for rIL-.beta., the most preferred process comprises suspending 
microbial cells having associated recombinant IL-1.beta. in an aqueous 
buffered medium having a pH from about 3.5 to about 4.5; disrupting the 
cells to extract solubilized IL-1.beta.; applying the solubilized 
IL-1.beta. to cation exchange media at a pH from about 2.5 to about 5.0; 
eluting the IL-1.beta. from the cation exchange media at a pH from about 
7.5 to about 9.0; applying the IL-1.beta. to anion exchange media in a 
buffer having low ionic strength; eluting the IL-1.beta. in a gradient of 
increasing salt concentration; applying the IL-1.beta. to media comprising 
pendant Procion Red dye ligand groups; and eluting the IL-1.beta. in a 
gradient of increasing sodium chloride concentration. 
1. Assays for IL-1 Activity and Endotoxin Levels 
Progress of rIL-1.alpha. or rIL-1.beta. purification can be monitored by a 
thymocyte mitogenesis assay, which involves ascertaining the capacity of a 
sample to induce proliferation of thymocytes from CD-1 mice. In this 
assay, approximately 1.times.10.sup.6 thymocytes, obtained from 10 to 12 
week old CD-1 mice (Charles River Breeding Laboratories, Wilmington, MA) 
are seeded in round bottom microplate wells (Corning Plastics, Corning, 
N.Y.) in the presence of three-fold serial dilutions of the IL-1 
containing fluid samples. The thymocytes are cultured in 150 .mu.l of 
Eagle's minimal essential medium (MEM) containing 50 U/ml penicillin, 50 
.mu.g/ml streptomycin, 2 mM glutamine, 0.2 mM gentamycin, 10 mM HEPES 
(N-2-Hydroxyethylpiperazine-N'-2-ETHANESULFONIC ACID) BUFFER, PH 7.4, 
TOGETHER WITH 4% w/v human serum and 10.sup.-5 M 2-mercaptoethanol. The 
samples are cultured for 72 hours at 37.degree. C. in an atmosphere of 5% 
CO.sub.2 in air. Thereafter, the cultures are pulsed for approximately 4 
hours with 0.5 microcuries (.mu.Ci) of tritiated thymidine (.sup.3 H-Tdr) 
after which the cultures are harvested onto glass fiber filter strips with 
the aid of a multiple-automated sample harvester. Details of this 
procedure are provided by Gillis et al., J. Immun. 120:2027 (1978) and in 
U.S. Pat. No. 4,411,992. 
In this assay, only the CD-1 cells cultured in the presence of IL-1 
incorporate .sup.3 H-Tdr in a dose-dependent manner. CD-1 cells cultured 
in the absence of IL-1 incorporate only background levels of radiolabel. 
IL-1 activity is calculated from the linear portion of the .sup.3 H-Tdr 
incorporation data. Units of IL-1 activity are determined as the 
reciprocal dilution of a sample which generates 50% of maximal thymocyte 
.sup.3 H-Tdr incorporation as compared to a laboratory standard. 
Alternatively, IL-1 activity can be assayed by an IL-1 conversion assay, 
which relies upon the discovery that IL-1 converts an IL-2 nonproducer 
cell line, the murine tumor cell line LBRM-33-145, to an IL-2 producer. In 
this assay, LBRM-33 -1A5 cells, (ATCC No. CRL-8079) are inactivated by 
addition of 50 .mu.g/ml mitomycin C and incubated for one hour at 
37.degree. C. 100 .mu.l of the inactivated cells (5.times.10.sup.5 
cells/ml) are cultured in 96-well flat-bottomed plates in the presence of 
an equal volume of the mitogen, phytohemagglutinin (PHA, 1%) together with 
serial dilutions of samples. At hourly time intervals the existence of 
IL-2 activity generated by IL-1 triggered, mitomycin C-inhibited 
LBRM-33-1A5 cells is directly ascertained by adding 50 .mu.l of IL-2 
dependent CTLL-2 cells (8.times.10.sup.4 cells/ml). The microwell cultures 
are then incubated for 20 additional hours followed by a 4 hour pulse 
with 0.5 .mu.Ci of .sup.3 H-Tdr, and the resulting pulsed cultures assayed 
for thymidine incorporation as detailed above. Only the CTLL-2 cells added 
to wells previously contacted with IL-1 (thereby inducing IL-2 production 
in the inactivated LBRM cells) will incorporate radiolabel. This 
conversion assay is both more rapid and more sensitive than the thymocyte 
mitogenesis assay. 
Protein concentrations can be determined by any suitable method. However, 
the Bio-rad total protein assay (Bio-rad Laboratories, Richmond, Calif. 
USA) is preferred. SDS-PAGE can also be employed to monitor purification 
progress, substantially as described by Kronheim et al., J. Exp. Med. 
161:490 (1985) or other suitable technique. Additional details regarding 
use of the IL-1 assays described above are disclosed by Conlon, J. Immun. 
131:1280 (1983) and Kronheim et al., supra. 
Endotoxin levels are conveniently assayed using a commercial kit available 
from Whittaker Bioproducts, Walkersville, Md., U.S.A., (Quantitative 
Chromogenic Lal QCL-1000) or its equivalent. This method uses a modified 
limulus amebocyte lysate and synthetic color-producing substrate to detect 
endotoxin chromogenically. Purified rIL-1.alpha. and rIL-1.beta. are 
tested for presence of endotoxin at multiple dilutions. The assay is 
preferably performed shortly following completion of purification and 
prior to storage at -70.degree. C. To minimize the possibility of 
bacterial contamination during the purification process itself, sterile 
buffers are preferably employed. 
2. Production of rIL-1.alpha. and rIL-1.beta. 
A. Construction of bacterial expression vectors 
Mature IL-1.alpha. and IL-1.beta. can be expressed in E. coliunder the 
control of the phage .lambda. PL promoter and cI857ts thermolabile 
repressor. Expression plasmids for rIL-1.alpha. and rIL-1.beta. production 
can be constructed from plasmid pPLc28 (ATCC 53082), plasmid pKK223-3 
(available commercially from Pharmacia Fine Chemicals, Uppsala, Sweden) 
and plasmids containing IL-1.alpha. clone 10A (March et al., supra; ATCC 
39997) and IL-1.beta. clone IL-1-14 (ATCC 39925) as follows. 
To create an expression vector for IL-1.alpha., a 3' portion of the 
IL-1.alpha. gene, extending from Ser.sup.113 (nucleotides 337-339) to 
Ala.sup.271 (nucleotides 811-813) is inserted into expression vector 
pPLc28. This is achieved by excising a 499 base pair AluI-NdeI fragment 
from the 10A clone, to which the following synthetic oligonucleotide 
linker is joined: 
##STR2## 
This linker includes AluI and EcoRI termini, a ribosome binding site, and 
ATG initiation codon in addition to the IL-1.alpha. Ser.sup.113 
-Ser.sup.117 sequence. pPLc28 is then digested to completion with EcoRI 
and NdeI, and the resulting larger fragment isolated by agarose gel 
electrophoresis. The linker, 10A clone, and plasmid fragments are then 
fused using T4 ligase, to provide an expression plasmid herein denoted 
pILP.alpha.. Additional details of the construction of pILP.alpha. can be 
found in the disclosure of copending, commonly assigned U.S. patent 
application Ser. No. 721,765, the disclosure of which is incorporated by 
reference herein. 
The resulting construct is then employed to transform E. coli strain 
.DELTA.H1 (ATCC 33767; Castellazi et al., Molec. gen. Genet. 117:211) to 
ampicillin resistance, using standard techniques. To express the 
plasmid-borne IL-1.alpha. gene, cultures of transformed .DELTA.H1 are 
grown in L-broth without ampicillin. When the cultures reach an A.sub.720 
of about 0.5, the culture temperature is raised to about 42.degree. C. to 
promote derepression of the thermolabile PL promoter. After one hour at 
elevated temperature, cells are harvested by centrifugation and 
flash-frozen in a dry-ice/methanol mixture. IL-1.alpha. activity in cell 
extracts can be assayed by either the thymocyte mitogenesis or IL-1 
conversion assays previously described. Details regarding purification 
procedures are provided in the following Examples. 
rIL-1.beta. can be produced via construction of an plasmid, herein 
designated pILP.beta.. This vector is assembled from pILPc (March et al., 
supra), which is constructed by replacing the BamHI/EcoRI fragment of 
pKK223-3 with a Sau3A/EcoRI fragment from pPLc28 containing the .lambda. 
PL promoter. This plasmid is digested to completion with EcoRI and PstI, 
and the largest fragment then ligated to a (1) a 669 base pair HpaII/PstI 
fragment from pIL-1-14 (ATCC 39925) containing the human IL-1.beta. gene 
(Ala117 to COOH terminus encodes active protein) and (2) the following 
EcoRI/HpaI synthetic oligonucleotide: 
##STR3## 
Plasmid pILP.beta. is then used to transform E. coli H1 or other cells 
containing a thermolabile repressor of PL transcription. Following growth 
to A.sub.720 of about 0.5, expression of the rIL-1.beta. gene is obtained 
by heat induction as previously described. rIL-1.beta. activity, as in the 
case of rIL-1.alpha., can be identified using the thymocyte mitogenesis or 
IL-1 conversion assays cited above.

EXAMPLES 1 AND 2: PROTEIN PURIFICATION 
The general purification scheme described in Examples 1 and 2, below, 
involved an initial acid extraction from cell pellets, followed by an SPS 
(Sulphopropyl Sephadex; Pharmacia) column chromatography step and elution 
from a DEAE-Sephacel (Pharmacia) column. Column fractions containing 
rIL-1.alpha. were then applied to Phenyl Sepharose CL-4B (Pharmacia), 
while those containing rIL-1.beta. were applied to a Procion Red agarose 
(Bethesda Research Laboratories) column for final purification. Sterile 
buffers were used throughout the purification protocol to safeguard the 
product from contamination by endotoxin. Chromatography fractions were 
monitored for protein concentration by the Bio-rad total protein assay 
(Bio-rad Laboratories, Richmond, Calif., USA) and the progress of 
purification evaluated by SDS-PAGE as described by Kronheim, J. Exp. Med. 
161:490 (1985). IL-1 activity of column fractions was determined by the 
IL-1 assays previously referenced. 
Experiments in which the pH of the initial extraction buffer was varied 
indicated that extraction of rIL-1.alpha. from E. coli cell suspensions at 
pH 2.8 resulted in precipitation of significant quantities of 
contaminating proteins while enabling good recovery of rIL-1.alpha.. 
Similar experiments involving rIL-1.beta. indicated that pH 3.9 is optimal 
for precipitating unwanted proteins while solubilizing rIL-1.beta.. Thus, 
the optimal pH for this initial extraction step may vary between fermenter 
batches. For this reason, small-scale pilot runs may be employed to 
determine optimal pH, particularly where large quantities of material are 
involved. 
rIL-1.alpha. and rIL-1.beta. were produced by growth and derepression of 
appropriate E. coli cells harboring high level thermolabile expression 
plasmids for rIL-1.alpha. and rIL-1.beta.. Cells were grown in a 10 liter 
Microferm Fermentor (New Brunswick) employing conditions of maximum 
aeration and vigorous agitation. An antifoaming agent (Antifoam A) was 
employed. Cultures were grown at 30.degree. C. in the super induction 
medium disclosed by Mott et al., Proc. Natl. Acad. Sci. USA 82:88 (1985) 
plus antibiotics, derepressed at a cell density corresponding to A.sub.600 
=0.05 by elevating the temperature to 42.degree. C. and harvested 16 hours 
after the upward temperature shift. The cell mass was initially 
concentrated using the Pellicon Cassette System (Millipore). The cell 
slurry was then centrifuged at 10,000 .times.g for 10 minutes at 4.degree. 
C. followed by rapid freezing of the cell pellets from 2.5 liter culture 
aliquots. 
To achieve the initial acid extraction, cell pellets obtained from 2.5 
liters fermentation medium as described above were suspended in about 20 
ml 30 mM Tris-HCl buffer, pH 8, containing 5 mM EDTA and 1 mM 
phenylmethylsulfonyl fluoride (PMSF). The resulting suspension was rapidly 
frozen in a dry ice/methanol bath and then thawed. Next, 200 ml of 30 mM 
sodium citrate buffer at pH 2.8 (rIL-1.alpha.) or 3.9 (rIL-1.beta.), 
containing 5 mM EDTA and 250 .mu.g/ml lysozyme was added to the 
suspensions. The resulting acid suspensions were incubated for 60 minutes 
in a 37.degree. C. water bath. Following incubation, the extracts were 
rapidly frozen in a dry-ice/methanol bath, thawed, and then centrifuged at 
4.degree. C. for 45 minutes at 38,000 .times.g. Supernatants were then 
carefully decanted for use in the next purification step. 
Extraction of rIL-1.alpha. from the E. colicell suspension at pH 2.8 
resulted in the precipitation of 79% of the contaminating proteins and 
recovery of 62.5% of the rIL-1.alpha.. The resulting extract was applied 
to an SPS C-25 column at pH 4. The column had been preconditioned with 
0.1% Triton X-100 (polyoxyethylene ether; Sigma Chemical Company, St. 
Louis, Mo., USA) and 10% fetal calf serum to reduce nonspecific absorption 
of IL-1 activity to the resin. First, the pH of the crude extract was 
raised to about 4.0 by addition of 1.0 N NaOH, and then the resulting 
solutions were applied to 20.times.2.5 cm columns containing SPS C-25, 
previously equilibrated with 10 mM sodium citrate, pH 4.0. The column was 
washed with 3 column volumes 10 mM 2-(N-morpholino)ethanesulfonic acid 
(MES) buffer, pH 5.0, and desired protein eluted from the column with 10 
mM Tris-HCl, pH 8.1. 10 ml fractions were collected, analyzed by SDS-PAGE, 
and stored at 4.degree. C. for additional purification. The elution with 
10 mM Tris-HCl buffer at pH 8.1 resulted in a pH rise after 3 column 
volumes and elution of the rIL-1.alpha., while 49% of the contaminating 
proteins remained bound to the gel. 
Fractions containing IL-1 activity from the previous step were combined and 
then applied to a 15.times.2.5 cm column containing DEAE-Sephacel 
previously equilibrated with 10 mM Tris-HCl pH 8.1. Since rIL-1.alpha. was 
eluted from the SPS column in the equilibration buffer of the DEAE column, 
the SPS pool (200-250 ml) could be loaded directly onto the DEAE column, 
avoiding loss of activity by dialysis. The DEAE column was washed with 
five column volumes of the starting buffer and then eluted with linear 
gradient of 0 to 600 mM NaCl in 10 mM Tris-HCl, pH 8.1, in a total of two 
column volumes. 5 ml fractions were collected, analyzed by SDS-PAGE, and 
held at 4.degree. C. for further purification. rIL-1.alpha. eluted from 
the column at 0.17-0.22 M NaCl. 66% of the contaminating proteins were 
eliminated in this step; 20% eluted earlier in the salt gradient and 46% 
remained bound to the gel after elution of rIL-1.alpha.. 
Fractions containing rIL-1.alpha. were pooled (50-60 ml) and treated by 
addition of sufficient solid ammonium sulfate to provide a final 
concentration of 0.5 M. The resulting solution was then applied to a 
30.times.2.5 cm column containing Phenyl Sepharose CL-4B, equilibrated 
with 10 mM Tris-HCl buffer, also 0.5 M in ammonium sulfate, at pH 8.1. The 
column was washed with 5 column volumes starting buffer, and eluted with a 
decreasing linear gradient of ammonium sulfate starting at 0.5 M and 
ending at 0 M in about 3 column volumes. Finally, the column was eluted 
with about 100 ml 10 mM Tris-HCl, pH 8.1. rIL-1.alpha. eluted at about 
0.25-0.10 M ammonium sulfate. 10 ml fractions were collected. PAGE of the 
fractions indicated that the Phenyl Sepharose step purified rIL-1.alpha. 
to homogeneity. Those fractions containing rIL-1.alpha. were pooled and 
concentrated by reapplication to SPS C-25 as described by Kronheim et al., 
supra. rIL- 1.alpha. was eluted using 10 mM phosphate buffered saline 
(PBS) at pH 8.2. 
Purified rIL-1.alpha. was stored at -70.degree. C. The purified 
rIL-1.alpha. contained only about 60 pg endotoxin per .mu.g IL-1.alpha., 
and exhibited a specific activity of 6.5.times.10.sup.8 units per mg. 
Overall yield for fermentation and purification was about 16.8 mg purified 
rIL-1.alpha. per liter culture. 
Extraction of rIL-1.beta. from the E. coli cell suspension at pH 3.9 
resulted in precipitation of 69% of the contaminating proteins and 
recovery of 37.3% of the rIL-1.beta. activity. Extracts containing 
rIL-1.beta. were also applied to an SPS C-25 column which had been 
pretreated with 0.1% Triton X-100 (polyoxyethylene ether; Sigma Chemical 
Company, St. Louis, Mo., USA) and 10% fetal calf serum. Protein was 
applied and eluted substantially as described for rIL-1.alpha., above. 
This SPS step removed 68% of the contaminating protein with good recovery 
of rIL-1.beta.. 
Fractions containing rIL-1 activity from the SPS step were combined and 
then applied to 15.times.2.5 cm columns containing DEAE-Sephacel 
previously equilibrated with 10 mM Tris-HCl pH 8.1. As described for 
rIL-1.alpha., above, the DEAE columns were washed with five column volumes 
of the starting buffer and then eluted with linear gradients of 0 to 400 
mM NaCl in a total of two column volumes. rIL-1.beta. eluted from the DEAE 
column at 0.075 M to 0.155 M NaCl, absent 62% of the contaminating 
proteins. 
Fractions containing rIL-1.beta. resulting from the DEAE column step were 
diluted 1:4 in 10 mM Tris-HCl buffer, pH 8.1, to reduce ionic strength to 
less than 40 mM, then applied to a 20.times.2.5 cm column containing 
Procion Red agarose previously equilibrated with 10 mM Tris-HCl buffer, pH 
8.1. The column was washed with five column volumes of starting buffer, 
and then eluted with a linear gradient in five column volumes ranging from 
0 to 1 M NaCl in 10 mM Tris-HCl buffer, pH 8.1. Fractions of 10 ml were 
collected, analyzed, and then concentrated as described for rIL-1.alpha., 
above. rIL-1.beta. eluted from the Procion Red column at 0.36-0.46 M NaCl. 
Purified rIL-1.beta. thus obtained exhibited a specific activity of 
1.95.times.10.sup.8 units per mg, and contained only 36 pg endotoxin per 
.mu.g rIL-1.beta.. Overall yield of protein was about 85.2 mg purified 
protein per liter culture. 
The results of the purification procedures described above are summarized 
in Table 1, below. 
TABLE 1 
______________________________________ 
Purification of Recombinant IL-1.alpha. and IL-1.beta. 
Total Specific 
Total Activity Activity 
Protein 
(U .times. 
Yield (U/mg 
(mg) 10.sup.10) 
(%) .times. 10.sup.7) 
______________________________________ 
A. IL-1.alpha.: 
Cell Suspension 
4576 8.0 100 1.7 
Cell Extract 968 5.0 62.5 5.1 
SPS Pool 493 5.0 62.5 5.1 
DEAE Sephacel pool 
168 2.45 30.6 15.0 
Phenyl Sepharose pool 
42 2.73 34.1 65.0 
B: IL-1.beta. 
Cell suspension 
10116 19.3 100 1.9 
Cell extract 3096 7.2 37.3 2.3 
SPS pool 978 13.2 68.7 13.5 
DEAE-Sephacel pool 
376 5.6 29.0 14.8 
Procion Red pool 
213 4.16 21.6 19.5 
______________________________________