Purification and Characterization of Human Recombinant Precursor ...

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Laboratories, King of Prussia, Pennsylvania 19406-0930. We have purified the .... at 37 "C and remain intact. At this stage in the purification! the protein was.

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

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

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 and stimulates a wide range of T-cell- and B-cell-dependent interleukin 18 (proIL18)fromrecombinant Esche- immune functions (2). Two cDNAs for IL1 have been cloned richia coli expressing the protein. The recombinant from both human and mouse (3-7) and have been shown to precursor was characterized by sodium dodecyl sul- correspond to the two known PI species of IL1, PI 5 and 7, fate-polyacrylamide gel electrophoresis, spectroscopy, ILla and ILlp, respectively (8, 9). Although ILla and ILlp Western blot, and for biological and receptor binding share only 25-30% amino acid similarity (10, ll), the two activity. The protein migrates at the expected molec- proteins are virtually indistinguishable in their spectrum of ular weighton sodiumdodecyl sulfate-polyacrylamide biological properties. ILla and ILlp compete for the same gel electrophoresis and analytical gel filtration col- receptor on all cells which have been examined to date (12). umns. The specific activity of the recombinant precur- Both ILla and ILlpare synthesized as precursor proteins sor is less than lo2units/mg in the EL4 thymoma assay of 31 kDaand aresubsequently cleaved to themature 17-kDa compared with 5 X 10’ unitslmg for the recombinant 17-kDa mature protein. The inactivity of the precursor form upon secretion (13-15). The function of the unusually large precursor region of these proteins is unclear; neither is attributable to the inability of the protein to bind the IL1 receptor on EL4 cells as shown by receptor com- encodes a typical hydrophobic leader sequence characteristic petition studies using ‘261-labeled 17-kDa ILla. Inac- of proteins which are secreted via the endoplasmic reticulum tivity of the ILla precursoris not due to degradation (3-5, 16). It is possible that this region is important for the secretion of IL1 by a yet undefined mechanism or that seof the protein in either the bioactivity or receptor binding assays. The inactive I L l s precursor is con- quences within the pre-region are responsible for other biological functions such as the intracellular accumulation of verted to an active form following proteolysis with a carboxyl-terminal precursor ILla and -p which has been observed inboth chymotrypsinwhichgenerates fragment of 17 kDa thatis 6 orders of magnitude more monocytes and monocytic cell lines (14,17-19). In any case, active than the starting ILla precursor. Removal of the degree of amino acid conservation within this region of the first 114 amino acids from proILla generates a the molecule is comparable to thatobserved within the mature fully active molecule. In contrast, removalof the first region of the six different ILl’s which have been sequenced.’ 77 amino acids by treatment with trypsin only parIt is, therefore, likely that the precursor region is critical to tially restores activity. The resultant 22-kDa protein the biological function of both ILla and ILlp. exhibits a600-foldincreaseinboth biological and The mature forms of ILla and ILlg have been purified receptor binding activity, demonstratinga direct cor- from recombinant sources (20-22) and extensively characterrelation between the ability of sequences within the ized (20, 21, 23, 24). In contrast, neither proILla or proILlP pro-region to inhibit biological activity and inhibit has been purified to homogeneity either from monocytes or binding to the IL1 receptor. Far-UV circular dichroism spectroscopy indicates that proILla is similar in sec- from recombinant Escherichia coli, and there are conflicting ondary structure to mature ILla; both proteins are reports with regard to the biological activity of the ILlP precursor (25-28). In this report we describe the purification nonhelicalsheetproteins. and initial biological and biochemical characterization of recombinant human ILlp precursor. The availability of homogeneous protein is crucial to the complete structural and functional characterization of the precursor and will also IL1’ is a cytokine produced by a wide variety of cell types in response to inflammation and injury (for a review see Ref. provide substrate for future studies on the processing and 1 and 2). IL1 is a central mediator of the immune response secretion mechanism of this cytokine. EXPERIMENTALPROCEDURES3 * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby RESULTS AND DISCUSSION marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Purification of Recombinant proILlp-We report on the 11 To whom correspondence should be addressed Dept. of Molecular Genetics, Smith Kline and French Laboratories, P.O. Box 1539, expression, purification, and initial biological and biochemical characterization of the recombinant human ILlp precursor. King of Prussia, PA 19406-0930. ‘The abbreviations used are: IL1, interleukin 1; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; proIL1, P. R. Young, unpublished work. precursor interleukin 1; r-proIL1, recombinant precursor IL1; PBS, Portions of this paper (including “Experimental Procedures” and phosphate-buffered saline; 2-ME, 2-mercaptoethanol; PMSF, phenyl- Figs. 1and 3-5) are presented in miniprint at theend of this paper. methylsulfonyl fluoride; PEG, polyethylene glycol; CHAPS, 3-[(3- Miniprint is easily read with the aid of a standard magnifying glass. cholamidopropyl)dimethylammonio]-l-2-hydroxy-l-propanesulfonicFull size photocopies are included in the microfilm edition of the acid. Journal thatis available from Waverly Press.

1689

1690

Precursor ILIB

Recombinant Human

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 frozen 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 thefinal product(35). This is thefirst studyin which the ILlp precursor has been isolated, and the purified protein used to characterize the biological and biochemical properties of the ILlpprecursor. 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 chromatography. It was necessary to chromatograph concentrated samples of proILlP in 1%2-mercaptoethanol to reduce intemolecular disulfides resulting in oligomerization of the protein. In the presence of reducing agent, proILlP eluted from acalibrated ACA54 column with anapparent molecular weight of 36,000 indicating that theprotein 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 theexpected molecular mass of 31 kDa on SDSPAGE (Fig. 2B, lane 4 ) . Specific Biologicaland Receptor Binding Activity of RecombinantproILlp-The specific activity of the purified rproILlp 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 precursor had a specific activity of lo2 units/mg, i.e. less than that of recombinant mature ILlp (Table I and Fig. 3, Miniprint). ProILlPdid not compete with '251-labeledmature

ILlp for binding to the IL1 receptor from EL4 cells at concentrations greater than lo' needed to achieve 50% competition 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 M, receptor was within the range of nonspecific binding ( Table I). The inactivity observed for proILlP in the EL4 assay was, therefore, fully explained by the inability of the protein to bind the IL1receptor. We determined the half-life of the precursor under conditions 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 thislevel of contamination is beyond our detection limit. In contrast to the datapresented 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 theprecursor 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 theexpression or isolation procedure, we attempted togenerate IL1 activity by treating the inactive precursor with trypsin and chymotrypsin. Trypsin treatmentgenerated a 22-kDa IL1 fragment (Fig. 6, lanes 2-4) and chymotrypsin resulted in a 17-kDa fragment which comigrated with mature ILlp onSDS-PAGE (Fig. 6, compare lunes 1 and 5-7). These proteolytically derived 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

A.

B. 1.

FIG. 2. Purification of recombinant precursor IL1/3. Recombinant precursor ILlp was purified as described under "Experimental Procedures" and is summarized in A. An aliquot of the sample a t each successive stage in the purification (1-4) was analyzed by SDSPAGE followed by Coomassie staining as shown in B. Lanes 1-4 correspond to stages 1-4 in A . The bandcorresponding to proILlPis indicated.

Lyse cells by thawing frozen cell pellet in hypotonic buffer ( lOmM Tris-HCI

pH7.0)

JA PO I5min 15.000 rpm

2 0-Sepharose

in I p m M Trts-HCI pH 7.0.0.1M NoCl Stepelute wlth 0.35M NaCl

3.

4.

1 1

Prectpltate with 38 5 % AmmoniumSulfate

Gel Filtration on A c A 5 4

* L '

*

Human Precursor Recombinant TABLE I Properties of precursor ILlp data card protease-generated products Molecular Specific N-terminal sequence

’ MAEVPKLASEMMAYYS ...

mass’

activity’

kDa 31 22 17 17

unitslmg

M

e102

>10-3

loooo

1691

I

Kd

MLVPCPQTFQENDLS ...‘ 6 X lo4 VHDAPVRSLNCTLRDS ...d 1 X 10’ ND ’ 5 X 10’ 10”’ APVRSLNCTLRDSQQK..: a Mobility on SDS-PAGE. EL4 assay as described under “Experimental Procedures.” Trypsin-generated fragment. Chymotrypsin-generated fragment. e Ref. 20.

76 114

ILlp

% J

-00051190

210 200

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 indicative of proteins which have predominantly p sheet structure. The absence of a strong peak a t 195 nm suggests little a-helical structure.

U

?!

2

Trypsin Chymotrypsin

Time (rnin)’5



No Addition

had a specific activity 6 orders of magnitude greater than proILlp, and within afactor of 5 of the specific activity 43.0 obtained for the recombinant mature ILlp protein. Given the variability of the bioassay and the error of determining the + proILlp concentration of protein by immunoblot and Coomassie stain25.7 0 ing, the specific activities of the 17-kDa proteolytically derived protein and the 17-kDa recombinant protein arecomparable. 18.4 The ability to generate afully active molecule from an inactive 0 precursor protein demonstrates that the lack of IL1 activity 14.3 is an intrinsic property of the ILlpprecursor. Therefore, the inactivity of the [email protected] cannot be attributed to arti6.2 0 facts of expression, isolation, or assay conditions. 3.0 e The 22-kDa fragment was 600-fold more active than the I 2 3 4 5 6 7 8 starting precursor in both the biological and receptor binding assays, but was 104-foldless active than mature ILlp; 6 X lo4 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, units/mg uersus 5 x 10’ units/mg. The presence of an addiand 15 min as indicated. Without treatment, proILlp isstable under tional 40 amino acids from the pro-region of ILlp was, therefore, sufficient to reduce the biological activity of this molethese conditions (lane 8). Mature ILlp (lane I ) andthetreated proILlp samples were electrophoresed on 15% SDS-PAGE, transcule by 10,000-fold relative to mature ILlp. Similar results ferred to nitrocellulose, and then probedwith anti-ILlp antisera were obtained when the precursor was treated with p l a ~ m i n . ~ which specifically reacts with the mature molecule. The 22-kDa fragment exhibited an affinity for the IL1receptor which was directly proportional to the relative biological sequence of proILlP and the mobility of these fragments on activity (Table I). Therefore, there is a direct correlation SDS-PAGE, the N termini of the chymotrypsin- and trypsin- between the ability of sequences from the pro-region of ILlp generated fragments probably correspond to amino acids 114 to inhibit biological activity and theability of these sequences and 77, respectively, i.e. these proteins contain an additional to prevent binding to the IL1receptor. 3 and 40 N-terminal amino acids compared toauthentic CD Spectroscopy of Recombinant proILlp-The decreased mature ILlp which begins a t amino acid residue 117 (Table biological and receptor binding activities of the various forms I). These fragments were stable to further proteolysis under of ILlp may result if a portionof the pro-region blocks access these conditions for several hours. No degradative products to the receptor binding site of the protein. Alternatively, the of proILl0 were observed under identical conditions in the amino acids required for receptor recognition may not be in absence of added protease (Fig. 6, lane 8). the configuration required for receptor binding, i.e. the proAnalysis of the resultant trypsin and chymotrypsin prod- teins may adopt different local or global conformations. To ucts by Coomassie staining indicated that the remaining N- examine potential conformational differences between preterminal portion of the precursor region was degraded (data cursor and mature ILlp, we compared the far-UV circular not shown), suggesting that theprecursor region may be less dichroism spectra of the two proteins. structurally compact and, therefore, more protease-accessible The circular dichroism spectrum of precursor ILlp is shown thanthe C-terminaldomain which comprises the active in Fig. 7. Analysis of the data for both precursor and mature moiety. Alternately, the N-terminaldomain may become un- ILlp using the Provencher algorithm (33, 34) indicated that stable once removed from the C-terminalsequences. Because despite the differences in activities, the two proteinsare the pro-region of IL1 is unusually long, it hasbeen speculated similar in their secondary structure. The crystal structure of that the N-terminal117 amino acids may contain additional the mature ILlp protein has been solved, and there is an activity(s). These results suggest that thepro-region of ILlp excellent correlation between the secondary structure preis labile. The intact pro-region fragment is therefore unlikely dicted by CD (20, 23) and that determined from the crystal to have a function other than as a component of the IL1 structure (24). CD spectra indicated that approximately 60% precursor. of the residues in mature ILlp occur in p sheets, with the The 22- and 17-kDa proteolytically generated proteinswere remainder in random coil (20, 23). Similarly, proILlP is 50% more active in the EL4 assay than the starting proILlp. The p sheet, 20% a helix, and 30% remainder. Therefore, both specific activities and receptor binding constants are comD. Hazuda, P. Simon, and P. Young, unpublished work. pared inTable I. The 17-kDa chymotrypsin-generated protein MW

P

15 30



5

15 30

l-5

30

Human Recombinant Precursor ILlp

1692

precursor and mature ILlp are nonhelical proteins composed primarily of p sheet secondary structure. Several observations suggest that themature andprecursor proteins may,however, adopt different tertiary conformations. The IL1precursor is unstable during isolation necessitating 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 inthe 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 binding activities. We cannot, however,exclude thealternate 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 published which reported similar experiments using partially purified ILlB precursor (35). The N-terminal sequences of the chymotrypsin-generated 17-kDa fragment which we deduced is confirmed by the N-terminal sequencing data presented inthat report. We also agree with the general descriptive trends of increases in biological activity with decreasing size of the pro-region which were reported. These investigators 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 provide 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 Livifor critical review of the manuscript.

8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

18.

19. 20. 21. 22. 23. 24. 25. 26. 27.

REFERENCES 1. Oppenheim, J. J., Kovacs, E., Matsushima, K., and Durum, S. K. (1986) Immunol. Today 7,45-56 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 315,641-647 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., Nomura, 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,

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. 1 6 4 , 237-250 Schmidt, J. A. (1984) J. Exp. Med. 1 6 0 , 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 Lymphokines (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. 134,343-349 Lumjuco, G., Galuska, S., Chin, J., Cameron, P., Boger, J., and Schmidt, J. A. (1986) Proc. Natl. Acad. Sci. U. S. A. 83,39723976 Hazuda, D. J., Lee, J. C., and Young, P. R. (1988) J. Biol. Chem. 263,8473-8479 Mizel, S. B. (1988) in Cellular and Molecular Aspects of Inflammation (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-LymphocyticCytokines (PoWanda, 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. 1 6 5 , 537-541 Wingfield, P., Payton, M., Taverneir, J., Barnes, M., Shaw, W., and Dayer, J.-M. (1986) Eur. J. Biochem. 1 6 0 , 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. 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., Hamerman, 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. 478,233-248 Laemmli, U. K. (1970) Nature 227,680-685 Simon, P. L., Laydon, J. T., and Lee, J. C. (1985) J. Immunol. Methods 84,85-94 Young, P. R., Hazuda, D. J., and Simon, P. L. (1988) J. Cell Biol. 107,447-456 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, 9437-9442

Human Recombinant Precursor ILlp

1693

SUPPLEMENTARV MATERIAL TO PURlFlCAnON AND CHARACTERIZAnON OF HUMAN PRECURSOR IL1 p

*..

RECEPTOR COMPETITION I

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