THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 264, No. 3, Isaue of January 25, pp. 1689-1693,1989 Printed in U.S.A.

Purification and Characterization of Human Recombinant Precursor Interleukin 1s”

(Received for publication, July 15, 1988)

Daria HazudaS, R. Lee Webb& Philip SimonV, and Peter YoungSII From the Departments of $Molecular Genetics, Wmmunology, and §Physical and Structural Chemistry, Smith Kline and French Laboratories, King of Prussia, Pennsylvania 19406-0930

We have purified the 31-kDa precursor of human interleukin 18 (proIL18) from recombinant Esche- richia coli expressing the protein. The recombinant precursor was characterized by sodium dodecyl sul- fate-polyacrylamide gel electrophoresis, spectroscopy, Western blot, and for biological and receptor binding activity. The protein migrates at the expected molec- ular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analytical gel filtration col- umns. The specific activity of the recombinant precur- sor is less than lo2 units/mg in the EL4 thymoma assay compared with 5 X 10’ unitslmg for the recombinant 17-kDa mature protein. The inactivity of the precursor is attributable to the inability of the protein to bind the IL1 receptor on EL4 cells as shown by receptor com- petition studies using ‘261-labeled 17-kDa ILla. Inac- tivity of the ILla precursor is not due to degradation of the protein in either the bioactivity or receptor binding assays. The inactive I L l s precursor is con- verted to an active form following proteolysis with chymotrypsin which generates a carboxyl-terminal fragment of 17 kDa that is 6 orders of magnitude more active than the starting ILla precursor. Removal of the first 114 amino acids from proILla generates a fully active molecule. In contrast, removal of the first 77 amino acids by treatment with trypsin only par- tially restores activity. The resultant 22-kDa protein exhibits a 600-fold increase in both biological and receptor binding activity, demonstrating a direct cor- relation between the ability of sequences within the pro-region to inhibit biological activity and inhibit binding to the IL1 receptor. Far-UV circular dichroism spectroscopy indicates that proILla is similar in sec- ondary structure to mature ILla; both proteins are nonhelical sheet proteins.

IL1’ is a cytokine produced by a wide variety of cell types in response to inflammation and injury (for a review see Ref. 1 and 2). IL1 is a central mediator of the immune response

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

11 To whom correspondence should be addressed Dept. of Molec- ular Genetics, Smith Kline and French Laboratories, P.O. Box 1539, King of Prussia, PA 19406-0930.

‘The abbreviations used are: IL1, interleukin 1; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; proIL1, precursor interleukin 1; r-proIL1, recombinant precursor IL1; PBS, phosphate-buffered saline; 2-ME, 2-mercaptoethanol; PMSF, phenyl- methylsulfonyl fluoride; PEG, polyethylene glycol; CHAPS, 3-[(3- cholamidopropyl)dimethylammonio]-l-2-hydroxy-l-propanesulfonic acid.

and stimulates a wide range of T-cell- and B-cell-dependent immune functions (2). Two cDNAs for IL1 have been cloned from both human and mouse (3-7) and have been shown to correspond to the two known PI species of IL1, PI 5 and 7, ILla and ILlp, respectively (8, 9). Although ILla and ILlp share only 25-30% amino acid similarity (10, ll), the two proteins are virtually indistinguishable in their spectrum of biological properties. ILla and ILlp compete for the same receptor on all cells which have been examined to date (12).

Both ILla and ILlp are synthesized as precursor proteins of 31 kDa and are subsequently cleaved to the mature 17-kDa form upon secretion (13-15). The function of the unusually large precursor region of these proteins is unclear; neither encodes a typical hydrophobic leader sequence characteristic of proteins which are secreted via the endoplasmic reticulum (3-5, 16). It is possible that this region is important for the secretion of IL1 by a yet undefined mechanism or that se- quences within the pre-region are responsible for other bio- logical functions such as the intracellular accumulation of precursor ILla and -p which has been observed in both monocytes and monocytic cell lines (14,17-19). In any case, the degree of amino acid conservation within this region of the molecule is comparable to that observed within the mature region of the six different ILl’s which have been sequenced.’ It is, therefore, likely that the precursor region is critical to the biological function of both ILla and ILlp.

The mature forms of ILla and ILlg have been purified from recombinant sources (20-22) and extensively character- ized (20, 21, 23, 24). In contrast, neither proILla or proILlP has been purified to homogeneity either from monocytes or from recombinant Escherichia coli, and there are conflicting reports with regard to the biological activity of the ILlP precursor (25-28). In this report we describe the purification and initial biological and biochemical characterization of re- combinant human ILlp precursor. The availability of homo- geneous protein is crucial to the complete structural and functional characterization of the precursor and will also provide substrate for future studies on the processing and secretion mechanism of this cytokine.

EXPERIMENTAL PROCEDURES3

RESULTS AND DISCUSSION

Purification of Recombinant proILlp-We report on the expression, purification, and initial biological and biochemical characterization of the recombinant human ILlp precursor.

P. R. Young, unpublished work. Portions of this paper (including “Experimental Procedures” and

Figs. 1 and 3-5) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

1689

1690 Human Recombinant Precursor ILIB

The identity of the recombinant precursor was confirmed by reactivity with ILlp-specific antisera by immunoblot (Fig. lB, Miniprint) and immunoprecipitation (15). Recombinant proILlP was extremely labile to proteolysis. By thawing fro- zen cell pellets in hypotonic buffer, the cells were rapidly lysed resulting in no detectable degradation of the solubilized protein (Fig. lB, lane 4, Miniprint). Lysis by other protocols, e.g. lysozyme or sonication, proved unacceptable due to a high degree of proteolysis. It was still necessary, however, to add an extensive mixture of protease inhibitors during the initial lysis step. A recent report in which partial purification of this protein was attempted did not undertake these precautions, resulting in multiple degradation fragments of the precursor which subsequently contaminated the final product (35). This is the first study in which the ILlp precursor has been isolated, and the purified protein used to characterize the biological and biochemical properties of the ILlp precursor.

The purification protocol summarized in Fig. 2 resulted in ILlp precursor which was greater than 90% pure and not contaminated by detectable degradation products. Following solubilization and fractionation on QAE-Sepharose (Fig. 2B, lane 2), proILlP could be incubated at 37 "C and remain intact. At this stage in the purification! the protein was virtually free of contaminating proteases. Ammonium sulfate precipitation effected a significant purification (Fig. 2B, lane 3 ) and concentrated the sample prior to gel filtration chro- matography. It was necessary to chromatograph concentrated samples of proILlP in 1% 2-mercaptoethanol to reduce inte- molecular disulfides resulting in oligomerization of the pro- tein. In the presence of reducing agent, proILlP eluted from a calibrated ACA54 column with an apparent molecular weight of 36,000 indicating that the protein is a monomer at physiological pH (data not shown). Overall yield of the final purified recombinant product was usually 50%. The purified product appeared as a single Coomassie-stained band and migrated at the expected molecular mass of 31 kDa on SDS- PAGE (Fig. 2B, lane 4).

Specific Biological and Receptor Binding Activity of Recom- binant proILlp-The specific activity of the purified r- proILlp was determined in a standard IL2 induction bioassay, the EL4 assay (31, Table I). In the EL4 assay, recombinant mature ILlp has a specific activity of 5 X 10' units/mg, closely approximating the value reported for the 17-kDa PI 7 species isolated from natural sources (20). The purified r-IL1p pre- cursor had a specific activity of lo2 units/mg, i.e. less than

that of recombinant mature ILlp (Table I and Fig. 3, Miniprint). ProILlP did not compete with '251-labeled mature

A.

FIG. 2. Purification of recombi- nant precursor IL1/3. Recombinant precursor ILlp was purified as described under "Experimental Procedures" and is summarized in A. An aliquot of the sam- ple a t each successive stage in the puri- fication (1-4) was analyzed by SDS- PAGE followed by Coomassie staining as shown in B. Lanes 1-4 correspond to stages 1-4 in A . The band corresponding to proILlP is indicated.

ILlp for binding to the IL1 receptor from EL4 cells at con- centrations greater than lo' needed to achieve 50% competi- tion using mature ILlp (Fig. 4, Miniprint). Mature ILlp exhibits a high affinity (KO = 10"' M ) for the receptor. Based on these numbers, the estimated Kd of proILlP binding to the receptor was within the range of nonspecific binding ( M, Table I). The inactivity observed for proILlP in the EL4 assay was, therefore, fully explained by the inability of the protein to bind the IL1 receptor.

We determined the half-life of the precursor under condi- tions of both the EL4 and soluble receptor assay to assure that the lack of receptor binding and biological activity was not due to degradation. Under these conditions the half-lives of both proILlP and mature ILlp (Fig. 5, A and B, Miniprint) were greater than 12 h. The inactivity of proILla was not a result of extensive degradation. Limited degradation could, however, account for the low level of specific activity (0.0001% of mature ILlp) observed for the precursor since processing of a small percent of the protein to a more active form may occur during assay, and this level of contamination is beyond our detection limit.

In contrast to the data presented here, other studies have claimed that the ILlp precursor is biologically active (26-28). However, since these studies failed to characterize either the relative amount or the molecular state of the protein, it was previously not possible to determine the specific activity of the precursor. Given the inherent sensitivity of proILlp to proteolysis, it is conceivable that some portion of proILlP was degraded to a more biologically active form(s). Another study in which precursor and mature ILlp were generated by in vitro translation demonstrated that the precursor had activity less than or equal to lo5 units/mg, the lower limit defined by their experimental conditions (25). Our results extend the lower limit of receptor binding and biological activity by three logs.

Activation of proILlp by Proteolysis-To determine if the inactivity of the r-proILlp was an intrinsic property of the precursor molecule and not an artifact of either the expression or isolation procedure, we attempted to generate IL1 activity by treating the inactive precursor with trypsin and chymo- trypsin. Trypsin treatment generated a 22-kDa IL1 fragment (Fig. 6, lanes 2-4) and chymotrypsin resulted in a 17-kDa fragment which comigrated with mature ILlp on SDS-PAGE (Fig. 6, compare lunes 1 and 5-7). These proteolytically de- rived products represent stable C-terminal domains of the protein which react with antiserum specific for the mature region of ILlp in Western analysis (Fig. 6). Based on the

B. 1. Lyse cells by thawing frozen cell

pellet in hypotonic buffer ( lOmM Tris-HCI p H 7 . 0 )

JA PO I5min 15.000 rpm

2 0-Sepharose in IpmM Trts-HCI pH 7.0.0.1 M NoCl Step elute wlth 0.35M NaCl

1 3. Prectpltate with 38 5 % Ammonium Sulfate

1 * L

' *

4. Gel Filtration on AcA54

Human Recombinant Precursor ILlp 1691

TABLE I Properties of precursor ILlp data card protease-generated products

N-terminal sequence Molecular Specific mass’ activity’ Kd

kDa unitslmg M ’ MAEVPKLASEMMAYYS ... 31 e102 >10-3 76 MLVPCPQTFQENDLS ...‘ 22 6 X lo4

114 VHDAPVRSLNCTLRDS ...d 17 1 X 10’ ND APVRSLNCTLRDSQQK..: 17 ’ 5 X 10’ 10”’

a Mobility on SDS-PAGE. EL4 assay as described under “Experimental Procedures.” Trypsin-generated fragment. Chymotrypsin-generated fragment.

e Ref. 20.

%

?! 2 Time

U J

Trypsin Chymotrypsin No Addition l-5

MW P ( rn in) ’5 15 30 ’ 5 15 30 ’ 30 43.0

+ proILlp

25.7 0

18.4 0

14.3

6 . 2 0

3.0 e

I 2 3 4 5 6 7 8

FIG. 6. Limited proteolysis of precursor ILlD. ProILlP (0.1 mg/ml) was treated with trypsin or chymotrypsin a t 37 “C for 5, 10, and 15 min as indicated. Without treatment, proILlp is stable under these conditions (lane 8). Mature ILlp (lane I ) and the treated proILlp samples were electrophoresed on 15% SDS-PAGE, trans- ferred to nitrocellulose, and then probed with anti-ILlp antisera which specifically reacts with the mature molecule.

sequence of proILlP and the mobility of these fragments on SDS-PAGE, the N termini of the chymotrypsin- and trypsin- generated fragments probably correspond to amino acids 114 and 77, respectively, i.e. these proteins contain an additional 3 and 40 N-terminal amino acids compared to authentic mature ILlp which begins a t amino acid residue 117 (Table I). These fragments were stable to further proteolysis under these conditions for several hours. No degradative products of proILl0 were observed under identical conditions in the absence of added protease (Fig. 6, lane 8).

Analysis of the resultant trypsin and chymotrypsin prod- ucts by Coomassie staining indicated that the remaining N- terminal portion of the precursor region was degraded (data not shown), suggesting that the precursor region may be less structurally compact and, therefore, more protease-accessible than the C-terminal domain which comprises the active moiety. Alternately, the N-terminal domain may become un- stable once removed from the C-terminal sequences. Because the pro-region of IL1 is unusually long, it has been speculated that the N-terminal 117 amino acids may contain additional activity(s). These results suggest that the pro-region of ILlp is labile. The intact pro-region fragment is therefore unlikely to have a function other than as a component of the IL1 precursor.

The 22- and 17-kDa proteolytically generated proteins were more active in the EL4 assay than the starting proILlp. The specific activities and receptor binding constants are com- pared in Table I. The 17-kDa chymotrypsin-generated protein

loooo I

-15000- 190 200 210 220 230 240

Wavelength (nm)

FIG. 7. Far-UV CD spectroscopy of precursor ILlD. The CD spectrum of proILl0 is shown from 190 to 240 nm. Conditions are as described under “Experimental Procedures.” The spectrum is indic- ative of proteins which have predominantly p sheet structure. The absence of a strong peak a t 195 nm suggests little a-helical structure.

had a specific activity 6 orders of magnitude greater than proILlp, and within a factor of 5 of the specific activity obtained for the recombinant mature ILlp protein. Given the variability of the bioassay and the error of determining the concentration of protein by immunoblot and Coomassie stain- ing, the specific activities of the 17-kDa proteolytically derived protein and the 17-kDa recombinant protein are comparable. The ability to generate a fully active molecule from an inactive precursor protein demonstrates that the lack of IL1 activity is an intrinsic property of the ILlp precursor. Therefore, the inactivity of the Ill@ precursor cannot be attributed to arti- facts of expression, isolation, or assay conditions.

The 22-kDa fragment was 600-fold more active than the starting precursor in both the biological and receptor binding assays, but was 104-fold less active than mature ILlp; 6 X lo4 units/mg uersus 5 x 10’ units/mg. The presence of an addi- tional 40 amino acids from the pro-region of ILlp was, there- fore, sufficient to reduce the biological activity of this mole- cule by 10,000-fold relative to mature ILlp. Similar results were obtained when the precursor was treated with p l a ~ m i n . ~ The 22-kDa fragment exhibited an affinity for the IL1 recep- tor which was directly proportional to the relative biological activity (Table I). Therefore, there is a direct correlation between the ability of sequences from the pro-region of ILlp to inhibit biological activity and the ability of these sequences to prevent binding to the IL1 receptor.

CD Spectroscopy of Recombinant proILlp-The decreased biological and receptor binding activities of the various forms of ILlp may result if a portion of the pro-region blocks access to the receptor binding site of the protein. Alternatively, the amino acids required for receptor recognition may not be in the configuration required for receptor binding, i.e. the pro- teins may adopt different local or global conformations. To examine potential conformational differences between pre- cursor and mature ILlp, we compared the far-UV circular dichroism spectra of the two proteins.

The circular dichroism spectrum of precursor ILlp is shown in Fig. 7. Analysis of the data for both precursor and mature ILlp using the Provencher algorithm (33, 34) indicated that despite the differences in activities, the two proteins are similar in their secondary structure. The crystal structure of the mature ILlp protein has been solved, and there is an excellent correlation between the secondary structure pre- dicted by CD (20, 23) and that determined from the crystal structure (24). CD spectra indicated that approximately 60% of the residues in mature ILlp occur in p sheets, with the remainder in random coil (20, 23). Similarly, proILlP is 50% p sheet, 20% a helix, and 30% remainder. Therefore, both

D. Hazuda, P. Simon, and P. Young, unpublished work.

1692 Human Recombinant Precursor ILlp

precursor and mature ILlp are nonhelical proteins composed primarily of p sheet secondary structure.

Several observations suggest that the mature and precursor proteins may, however, adopt different tertiary conforma- tions. The IL1 precursor is unstable during isolation necessi- tating the addition of protease inhibitors and lysis conditions not required for the purification of recombinant mature ILlB (20). In mature ILlB, the two free sulfhydryls are buried (20, 24), and intermolecular disulfides do not form spontaneously unless the protein is denatured (23). Like the denatured mature ILlp, the precursor aggregated in the absence of reductant. The observation that the precursor may adopt a more open tertiary conformation than the mature protein would explain the differences in biological and receptor bind- ing activities. We cannot, however, exclude the alternate hypothesis that the pro-region excludes binding by imposing steric constraints on the receptor binding site of the protein. The availability of purified ILlp precursor will allow us to address these and other issues regarding the biology of ILlB.

After these studies had been completed, a paper was pub- lished which reported similar experiments using partially purified ILlB precursor (35). The N-terminal sequences of the chymotrypsin-generated 17-kDa fragment which we de- duced is confirmed by the N-terminal sequencing data pre- sented in that report. We also agree with the general descrip- tive trends of increases in biological activity with decreasing size of the pro-region which were reported. These investiga- tors could not establish a lower base-line level for the inactive ILlp precursor because of contaminating lower molecular weight degradation products. Their studies also did not pro- vide sufficient data to calculate specific activities for either the precursor or the proteolytically processed forms of proILl(3 which are essential for comparison with the activity of mature ILlp as well as for delineating which region(s) is responsible for down-modulating the activity of the precursor. In contrast, our results using the purified ILlp precursor are quantitative and provide comparative data on the specific activity as well as K d values for the various forms of the protein.

Acknowledgments-We would like to thank Jay Lillquist, Randy Deck, and William Fenderson for excellent technical assistance, and Chester Meyers and George Livi for critical review of the manuscript.

REFERENCES 1. Oppenheim, J. J., Kovacs, E., Matsushima, K., and Durum, S. K.

2. Dinarello, C. (1988) FASEB J. 2 , 108-115 3. March, C. J., Mosley, B., Larsen, A., Cerretti, D. P., Braedt, G.,

Price, V., Gillis, S., Henney, C. S., Kronheim, S. R., Grabstein, K., Conlon, P. J., Hopp, T. P., and Cosman, D. (1985) Nature

4. Auron, P. E., Webb, A. C., Rosenwasser, L. J., Mucci, S. F., Rich, A., Wolff, S. M., and Dinarello, C. A. (1984) Proc. Natl. Acad. Sci. U. S. A. 8 1 , 7907-7911

5. Gray, P. W., Glaister, D., Chen, E., Goeddel, D. V., and Pennica, D. (1986) J. Zmmunol. 137,3644-3648

6. Furutani, Y., Notake, M., Yamayoshi, M., Yamagishi, J.-I., No- mura, H., Ohue, M., Ruruta, R., Fukui, T., Yamada, M., and Nakamura, S. (1985) Nucleic Acids Res. 13,5869-5882

7. Lomedico, P. T., Gubler, U., Hellman, C. P., Dukovich, M., Giri,

(1986) Immunol. Today 7,45-56

315,641-647

8.

9. 10.

11.

12. 13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30. 31.

32.

33. 34.

35.

J. G., Pan, Y.-C. E., Collier, K., Semionow, R., Chau, A. O., and Mizel, S. (1984) Nature 312,458-462

Cameron, P. M., Limjuco, G., Chin, J., Silberstein, L., and Schmidt, J. A. (1986) J. Exp. Med. 164 , 237-250

Schmidt, J. A. (1984) J. Exp. Med. 160 , 772-787 Auron, P. E., Rosenwasser, L. L., Matsushima, K., Copeland, T.,

Dinarello, C. A., Oppenheim, J. J., and Webb, A. C. (1985) J. Mol. Cell. Immunol. 2 , 169-177

Lomedico, P. T., Gubler, U., and Mizel, S. B. (1987) in Lympho- kines (Pick, E., ed) Vol. 13, pp. 139-150, Academic Press, New York

Dower, S. K., and Urdal, D. L. (1987) Immunol. Today 8, 46-51 Giri, J. G., Lomedico, P. T., and Mizel, S. B. (1985) J. Immunol.

Lumjuco, G., Galuska, S., Chin, J., Cameron, P., Boger, J., and Schmidt, J. A. (1986) Proc. Natl. Acad. Sci. U. S. A. 83,3972- 3976

Hazuda, D. J., Lee, J. C., and Young, P. R. (1988) J. Biol. Chem.

Mizel, S. B. (1988) in Cellular and Molecular Aspects of Inflam- mation (Poste, G., and Crooke, S. T., eds) pp. 75-93, Plenum Publishing Corp., New York

Young, P. R., Hazuda, D. J., Connor, J. R., and Dalton, B. (1988) in Monokines and Other Non-Lymphocytic Cytokines (Po- Wanda, M., Oppenheim, J. J., Kluger, M. J., and Dinarello, C. A., eds) pp. 83-88, Alan R. Liss, Inc., New York

Auron, P. E., Warner, S. J. C., Webb, A. C., Cannon, J. G., Bernheim, H. A., McAdam, K. J. P. W., Rosenwasser, L. J., LoPreste, G., Mucci, S. F., and Dinarello, C. A. (1987) J. Immunol. 138,1447-1456

Singer, I. I., Scott, S., Hall, G. L., Limjuco, G., Chin, J., and Schmidt, J. A. (1988) J. Exp. Med. 167,389-407

Meyers, C. A., Johansen, K. O., Miles, L. M., McDevitt, P. J., Simon, P. L., Webb, R. L., Chen, M.-J., Holskin, B. P., Lillqist, J. S., and Young, P. R. (1987) J. Biol. Chem. 262,11176-11181

Wingfield, P., Payton, M., Graber, P., Rose, K., Dayer, J.-M., Shaw, A. R., and Schmeissner, U. (1987) Eur. J. Biochem. 165 ,

Wingfield, P., Payton, M., Taverneir, J., Barnes, M., Shaw, W., and Dayer, J.-M. (1986) Eur. J. Biochem. 160 , 491-497

Craig, S., Schmeissner, U., Wingfield, P., and Pain, R. H. (1987) Biochemistry 26, 3570-3576

Priestle, J . P., Schar, H.-P., and Grutter, M. G. (1988) EMBO J. 7,339-343

Mosley, B., Urdal, D. L., Prickett, K. S., Larsen, A., Cosman, D., Conlon, P. J.. Gillis, S.. and Dower, S. K. (1987) J. Biol. Chem.

134,343-349

263,8473-8479

537-541

262,2941-2944 . .

Auron. P. E.. and Webb. A. C. (1987) in Lvmwhokines (Pick. E.. . , . ed) Vol. 14, pp. 33-58,'Academic Press, New York

Wood, D. D., Bayne, E. K., Goldring, M. B., Gowen, M., Ham- erman, D., Humes, J. L., Ihrie, E. J., Lipsky, P. E., and Staruch, M.-J. (1985) J. Immunol. 134,895-903

Kimball, E. S., Simon, P. L., Saklatvala, J., Metzner, R., and Wood, D. D. (1986) Lymphokine Res. 5,119-125

Shatzman, A. R., and Rosenberg, M. (1986) Ann. N. Y. Acad. Sci.

Laemmli, U. K. (1970) Nature 227,680-685 Simon, P. L., Laydon, J. T., and Lee, J. C. (1985) J. Immunol.

Young, P. R., Hazuda, D. J., and Simon, P. L. (1988) J. Cell Biol.

Provencher, S. W. (1982) Comput. Phys. Commun. 2 7 , 229-242 Provencher, S. W., and Gloeckner, J. (1981) Biochemistry 2 0 ,

33-37 Black, R. A., Kronheim, S. R., Cantrell, M., Deeley, M. C., March,

C. J., Prickett, K. S., Wignall, J., Conlon, P. J , Cosman, D., Hopp, T. P., and Mochizuki, D. Y. (1988) J. Biol. Chem. 263,

478,233-248

Methods 84,85-94

107,447-456

9437-9442

Human Recombinant Precursor ILlp 1693 SUPPLEMENTARV MATERIAL

TO PURlFlCAnON AND CHARACTERIZAnON OF

HUMAN PRECURSOR IL1 p *..

RECEPTOR COMPETITION I

\