Feb 3, 1987 - Johanson, Lynette M. Miles, Patrick J. McDevitt, Philip L. Simon,. R. Lee Webb, Mann-Jy Chen, Beverly P. Holskin, Jay S. Lillquist, and Peter R.
THEJOURNAL OF
Vol. 262, No , 23, Issue of August 15, pp. 11176-111S1,1987 Printed in U.S.A.
BlOLOGtCAL CHEMISTRY
0 1987 by The American Society for Biochemistry and Molecular Biology, Inc.
Purification and Characterization ofHuman Recombinant Interleukin- lB* (Received for publication, February 3, 1987)
Chester A. Meyers, Kyung 0. Johanson, LynetteM. Miles, PatrickJ. McDevitt, PhilipL. Simon, R. Lee Webb, Mann-Jy Chen,Beverly P. Holskin, Jay S. Lillquist, and Peter R. Young From the Departments of Macromkcular Sciences, Protein Biochemistry, and Molecular Genetics, Smith Kline and French Laboratories; Swedeland; Pennsylvania 19479
A human interleukin-1 (IL-1) cDNA was cloned, 1p in accordance with March et al. (5). and the region coding for the mature proteinwas exWhile the purification and partial characterizationof minpressed in Escherichia coli. The 17-kDa biologically ute quantities of IL-1 from natural sources has been reported active product was purified in 40%yield to apparent @-lo), the physicochemical properties of purified recombihomogeneity,withoutchaotropes,fromthesoluble nant IL-1 have not been extensively described. Gubler et al. fraction of sonicatedcell lysates. The recombinant IL- (11)recently published a purification method for a nearly full 18 was characterizedby aminoacid analysis, NH2- and length human rIL-la expressed in Escherichia coli, although COOH-terminal sequence analysis, sodium dodecyl sul- they noted a 20-fold loss in biological activity as aconsequence fate-polyacrylamidegel electrophoresis, spectroscopy, of using chaotrope-containing buffers. We report here an and biological assay. Specific biological activity was 4.6 x 10’ unitslmg in a co-mitogenic IL-2 induction efficient purification scheme for our recombinant human ILassay using cultured EL-4 T-lymphocytes. The molar l p starting from the soluble cell lysates of an E. coli expression extinction coefficient was determinedtobe 10,300 system, and we provide chemical and physical characterizacm” M” at 280 nm. NH2-terminal sequence analysis tion of the fully biologically active product. A preliminary revealed that 70%of the product begins with the Ala report of this work has been presented (12). The availability of hundreds of milligrams of purified recombinant IL-lP will corresponding to the NH2 terminus of the natural protein, while 30% begins with the following Pro. No permit extensive structural studies tobe undertaken, as well as help to clarify and confirm the multiple biological activities, initiator Met was observed.Bothofthesulfhydryl groups are reactive to Ellman’s reagent and to iodoa- mechanisms of action, and physiological significance of this class of immunoregulatory molecules. cetamideundernonreducingconditions,indicating that the Cys residues do not form disulfide bonds. SCarboxamidomethyl-Cys-rIL18 retainedbiological EXPERIMENTAL PROCEDURES~ activity in theIL-2 induction assay. Circular dichroism suggested anextensive 8 sheet structure forrIL-1s. RESULTS Expression of IL-I@in E. coli Interleukin-1 (IL-1)’is a low molecular weight protein with a broad range of biological activities, including lymphocyte activation ( l ) , fever induction (2), stimulationof acute phase hepatic responses (3), andstimulation of collagenase and prostaglandin production (4). Several forms of IL-1 have been identified which exhibit molecular weight and charge heterogeneity. Recently, two distinct human DNAs were isolated from an activated macrophage cDNA library, and although distantly related, were shown to encode proteins with IL-1 activity (5). One cDNA sequence showedhomology to the sequence reported by Lomedico et al. (6) for murine IL-1, while the other was almost identical to the human sequence described by Auron et al. (7). March et al. (5) distinguished between the separate genes as IL-1 a and IL-1 p. The active maturehumanproteins, each apparently processed from larger (31 kDa) precursors to about 17 kDa, are 159 and 153 residues long and are respectively designated IL-la and IL-
Fig. 1 shows the human IL-lp expression vector. Based on the Western blot (Fig. 2), approximately 80% of the rIL-lp was soluble upon lysis of the bacteria in buffers which do not contain detergents or chaotropic agents, and in this fraction it constituted approximately 5% of total E. coli protein as judged by Coomassie staining. When the crude soluble fraction was tested in the EL-4 co-mitogen assay it exhibited high biological activity (3-5 X lo6 units/ml), consistent with the estimated IL-1p protein concentration and published specific activities for natural IL-lp. A sample whichwas partially purified by preparative SDS-PAGE and recovered by electroelution had the expected amino acid composition, and it still retained residual activity in the EL-4 assay; however, amino-terminal sequence analysis indicated two forms, the major one beginning Ala-Pro-Val-, identical to the natural protein (5,8,25), and theminor form beginning Pro-Val-. No initiator methionine was detected.
* 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. The abbreviations used are: IL, interleukin; SDS, sodium dodecyl sulfate; BSA, bovine serum albumin; PAGE, polyacrylamide gel electrophoresis; PMSF, phenylmethylsulfonyl fluoride; PTH, phenylthiohydantoin; HPLC,high performance liquid chromatography; dansyl, 5-dimethylaminonaphthalene-1 sulfonyl; CM, carboxymethyl.
* Portions of this paper (including “Experimental Procedures,” Tables I1 and 111, and Figs. 3-7) 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 available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 87 “320, cite the authors, and include a check or money order for $6.80 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.
11176
Human Recombinant Interleukin-lp Purification and Characterization of rIL-18 The chaotrope-free purification of recombinant human IL18 from E. coli is summarized in Table I. The use of a Sephadex G-25 column for desalting prior to S-Sepharose chromatography (Fig. 3, Miniprint) avoids the losses in product and time experienced with dialysis. Partial purification was also achieved at this step as judged by SDS-PAGE of individual column fraction samples.Based on the specific activity, the protein was judged to be nearly homogeneous after S-Sepharose chromatography (Table I).Residual higher molecular weight contaminants were removed as shown in NdeI
ILlBcDNA
HpoI
x
( A d )
11177
Fig. 4 (Miniprint). Overall yields of final product were typically 40% based on total unitsof IL-1 activity in the starting cell lysate supernatants. No losses in biological activity or changes in SDS-PAGE behavior were detected during storage at 4 "C in the final buffer at 1 mg/ml for 4 weeks in the pH range 4-9 (data not shown).
Specific Activity The specific activity of 4.6 x 10' units/mg compares favorably to the specific activity reported for IL-18 isolated from natural sources (see "Discussion"). We also tested the rIL-18 in the mouse thymocyte assay (26) for a more direct comparison to previously reported activities. Our rIL-1/3had a specific activity of1.6 X lo7 units/mg in that assay, in excellent agreement with the value of 1.7 X lo7 units/mg determined for natural IL-18(8). SDS-PAGE The purified rIL-10 appears as a single Coomassie-stained band at the expected mass of 17.5 kDa at loads up to 40 pg (Fig. 5, Miniprint). Silver staining produced identical results for up to 10-pgloads (not shown). The final product migrates identically with the corresponding band that was demonstrably induced in theE. coli lysates and is estimated (Table I)to comprise approximately 5% of total protein.
u pMGILlB
AmpR
FIG.1. Human IL-lDexpression vector. Shoded region represents the mature 17 kDa human IL-l&coding region consisting of a Amino Acid Analysis synthetic oligonucleotide linker (sequence is shown) connected to a HpaII/AccI restriction fragment derived from the IL-la cDNA (5). The average composition from duplicate analyses of three Circled nucleotides arethose altered fromthe cDNA sequence either samples of rIL-l/3 is shown in Table I1 (Miniprint). Values to create the PvuI site or to provide E. coli-preferred codon usage.
"+ - + AR13
are uncorrected for losses during hydrolysis, yet with the usual exception of Ser, all the amino acids are within 10% of the amount predicted from the cDNA sequence (5,7).
AR68 AR58
+ -
Determination of Molar Extinction Coefficient Quantitative amino acid composition and spectral analyses at pH 7.5 were performed in order to determine a molar - 43 extinction coefficient for rIL-18. For the calculation based on amino acid analysis (Table 11, Miniprint), the protein concen- 25.7 tration was determined by dividing the observed molar con- 18.8 centration of leucine byits predicted value of 15 residues/mol rILl* - 14.3 of protein. This method gave a calculated extinction coefficient of 10.1 X lo3 cm" at 280nm. A 1 mg/ml solution - 6.2 of rIL-18 has an AZWnmof0.58. These values are in close agreement with an tmnmof 10.3 X lo3cm" M-' and an AzWnm FIG.2. Western immunoblot analysis of IL-18synthesized of 0.59 for a 1mg/ml solution, which we calculated from the AR58, AR68) of E. coli. E. coli lysate predicted spectrophotometric contributions of Trp and Tyr in three strains (AR13, supernatant ( S ) and pellet (P)fractions are shown. Sample loads for according to Edelhoch (27) using an absorbance correction differentstrainsarenotequivalent.Autoradiographyshowsthat factor determined as described previously (28).
SP S P S P S P S P S P
following thermal induction of the plasmid, IL-lD is recognized by the antibody in hosts containing pMGILl@(+), but not in controls (-) which are either host alone (AR13), or host with control vector pMG27M- (AM8 and AR68). Molecular weight standards are indicated.
TABLE I Purification of Human rIL-l@ Specific
activity" Fraction
Total
Total
units X IO-"
mg
activity unitslmg X IO-'
NHz-Terminal Sequence Analysis Table I11 (Miniprint) shows the results obtained from the first 20 cycles of automated Edman sequence analysis of purified rIL-18. The first cycle contained no detectable Met, which is the initiator amino acid for E. coli expression. Only Ala and Pro were detected in cycle 1, and two signals were present in each cycle where positive identification of both residues could be made. In those cycles where both residues could be reliablyquantitated, the amino acids were present in an average ratio of about 2.51, and were in each case consistent with an identification of the major species as mature human rIL-10, and theother as des-[Ala']-IL-1/3.
0.35 13,0Wb 4.57 Lysate supernatant 9,800b 0.41 Ammonium sulfate 4.02 4.25 710' 3.02 S-Sepharose 4.82 524' 2.53 Sephadex G-50 4.60 436' 2.0 DEAE-Sephacel COOH-Terminal Sequence Analysis 1 unit/ml produces half-maximal activity for IL-1 the in EL-4 TFig. 6 (Miniprint) shows the time course of release of amino lymphoma assay. acids from the COOH terminus of human rIL-18 by the action Calculated bythe method of Bradford(34). of carboxypeptidase Y. The number of picomoles of each Calculated from the Am,.
11178
HumanInterleukin-1 Recombinant
0
(Table 11); and ( d ) NH2-terminal sequence analysis (Table 111). The specific activity also compared well with that reported for the protein isolated from cultured mononuclear cells. Simon et al. (24) have shown that the EL-4 assay is about one order of magnitude more sensitive than themurine thymocyte co-mitogenic assay which wasused to measure the specific activity reported for IL-16 purified from natural sources (8).Our value of 4.6 X 10' EL-4-units/mg for rIL-16 is consistent with that observation. We also tested our recomNative Molecular WeightDetermination of rIL-16 binant IL-16 in the thymocyte co-mitogenic assay in order to The rIL-lpeluted from a calibrated Superose-12 column a t obtain a more direct comparison with published values for an apparent molecular weight of 18,700. This indicates that specific activity than could be expected using the EL-4assay. The result was in excellent agreement with the literature the protein exists as a monomer a t a physiological pH. value (8). In the first 20 cycles of NHP-terminal sequence analysis of Analysis of Sulfhydryl Groups rIL-16, we unambiguously identified 18 residues which Ellman Analysis-No reaction occurred in Buffer 1 during a 4-h incubation period, while the reaction in Buffer 2 was matched the predicted sequence of mature IL-10. Heterogeneity at the NH2 terminus was revealed by the presence in instantaneous and showed 1.94 molSH/mol of protein. each cycle of two signals, one of which corresponded to the Reaction with Iodoacetamide-2.02 mol of CM-Cys/mol of sequence protein were found by amino acid analysis of the dialyzed expected mature proteinsequence, and the other ato which lacked the NHz-terminal Ala residue, but was otherwise reaction product. The specific activity of the carboxamidomethylated protein was 3.1 x 10' unitsjmg which was approxi- identical to the mature IL-16 sequence. The truncated promately 50% of the specific activity measured for control tein, des-[Ala']-IL-l@, comprised approximately 30% of the total IL-16 preparation. Similar results were obtained with samples of rIL-16 in the same EL-4assay. Circular Dichroism Spectra-The results of circular dichro- the lysate material electroeluted from a gel, indicating that the NH2-terminal processing probably did not occur during ism spectral analyses are given in Fig. 7 (Miniprint). our purification steps. Preliminary studieson mixtures of the two forms, which partially separated during anion exchange DISCUSSION chromatography, indicated no discernible differences in speThis report describes our cloning of the human JL-16 cDNA cific biologicalactivity (EL-4 assay). Therewas no detectable expression of the mature form of IL-lp protein in E. coli and NHz-terminalinitiator methionine observed in the rIL-16 an efficient method for purifying hundreds of milligrams of preparations. In contrast, other E. coli-derived recombinant the biologically active recombinant product. The availability proteins such as human interferon (Y (29), interferon y (30), of such quantities has permitted extensive physicochemical and growth hormone (31), often retain the methionyl NH,characterization of this protein. terminal extension of the mature protein sequence. Recently, Following cell lysis, about 80% of the protein was found in heterogeneity at the NH, terminus of recombinant human the soluble fraction of the cell lysis buffer, so that thepurifiIL-2 produced in E. coli was observed such that 75% of the cation steps could be performed at neutral pH without the protein began with Met, while the remaining 25% began with use of chaotropic agents. Previous reports of IL-1 expression Ala' of the mature protein (32). The extent to which postin E. coli have included the use of chaotropes for solubilization translational NH,-terminal processing occurs in recombinant of the product followingcell lysis. Indeed, many proteins human IL-16 and IL-2 appears different, despite the fact that expressed at high levels in E. coli appear in inclusion bodies they sharean Ala-Pro sequence following the initiator methiand areconsequently found in the insoluble fraction upon cell lysis. The presence of chaotropic agents during the purifica- onine. Although the conditions which give rise to NH2 terminally modified recombinant proteinsarenot clear, the tion of recombinant human IL-la was associated with at least its occurrence underscore the general frequent examples of a 20-fold decrease in its biological activity (11), which emphasizes the general desirability of avoiding denaturing condi- need for extensive structural characterizationof recombinant tions. We have, however, observed that purified rIL-16 ex- protein products to detect possible differences from the preposed to 6 M guanidine for 24 h regains full biological activity dicted structures. Further evidence of correct structure was obtained by in the EL-4 mitogenic assay upon removal of the chaotrope COOH-terminal sequence analysis (Fig. 6) which confirmed by dialysis (data not shown). As shown in Table I, the most effective single step in the that the IL-16 is indeed full length and has the predicted purification scheme is S-Sepharose chromatography which, COOH-terminal amino acid sequence. Internal sequences, based on the specific activity, provides nearly pure product in verified by reverse phase HPLC separation of CNBr-treated a single step. SDS-PAGE and amino acid analysis further rIL-16 (not shown), matched the results published by Camindicated substantial purity (>go%)at this step (notshown). eron et al. (8) for CNBr-treated natural human IL-16. We were interested in determining whether the two halfThis was achieved following our observation that at an optimal buffer pH of 5.2 most of the contaminating host proteins cystine residues had formed disulfides, and whether Cys is were not retained on the column in the low ionic strength required for biological activity. The results of the Ellman conditions required to bind the rIL-16. No product loss was experiment, where both Cys residues were reactive under detected duringthe extensive washing that was usedto remove nonreducing conditions, indicate that the recombinant protein does not readily form disulfide bonds during its isolation unbound proteins prior to elution of the IL-16. High purity of the final product was indicated by the or storage. The behavior of rIL-16 on a calibrated Superosefollowing: (a) constantspecific activity in the last three puri- 12 column at pH 8 indicates that the protein existed as a fication steps (Table I); ( b ) SDS-polyacrylamide gel electro- monomer, in further support that the rIL-16 is resistant to phoresis (Fig. 5 ) ; ( c ) good correlation of the amino acid spontaneous disulfide bond formation. It is noteworthy that the cysteine sulfhydryl groups failed to react with Ellman's analysis with the predicted composition of naturalIL-16 residue released at each time point represents the observed yield from 60 pmol of starting IL-16 protein. The nearly 2fold recovery of Ser is consistent with the presence of a SerSer sequence expected at theextreme COOH terminus of IL16. The recovery of Ser at 60 min (not shown) was in fact quantitative (119.5 pmol). A total of 7 amino acid residues were released in the correct order for the predicted COOHterminal sequence of IL-16.
Human Recombinant Interleukin-l@ reagent in the absence of chaotrope, suggesting that the RSH moieties are inaccessible or “buried” within the molecule. Carboxamidomethyl-Cys-rIL-l/3 retained substantial biological activity in the EL-4 assay, despite the loss of any ability to form disulfides in the course of the assay. This demonstrates a lack of requirement for disulfides or free sulfhydryl groups in the biological action of IL-lP, at least in the IL-2 induction assay. This result is not surprising since human ILla has only 1 Cys residue (5) and murine IL-1 has none (6). The availability of a large amount of purified human rILI@ has made possible the determination of an extinction coefficient, circular dichroism spectral analysis, and chemical modification which yielded information about the role of Cys in the biological action of this protein. Many of the properties attributed to IL-1 can now be retested with the pure recombinant product, receptor studies can be extended, and sufficient materialis available for fragmentation andmodification studies to further elucidate structure-function relationships in this importantclass of immunomodulators. As this paperwas completed, Kronheim et al. (33) published a dissimilar purification method for their recombinant IL-la, and they presented evidence of similar but more extensive processing at the NH2 terminus. There was good agreement of specific biological activities in comparable assays (33). However, their product was reported to have an anomalously low PI of 5.5 rather than the expected PI of 7 that has been determined for the natural protein (21). We have determined the PI of our recombinant IL-lP to be 6.9 by isoelectric focusing and 7.0 by chromatof~cusing.~ Acknowledgments-We are grateful to Dr. James Strickler for valuable help and advice with the protein sequencing studies and to Dr. Ganesh Sathe for providing the synthetic oligonucleotides.
REFERENCES 1. Mizel, S. B. (1982) Zmmunol. Reu. 6 3 , 51-72 2. Murphy, P. A., Simon, P. L., and Willoughby, W. F. (1980) J. Immunol. 124,2498-2501 3. Dinarello, C. A. (1984) Rev. Infect. Db.6, 51-95 4. Mizel, S. B., Dayer, J-M., Krane, S. M., and Mergenhagen, S. E. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 2474-2477 5. 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 6. Lomedico, P. T., Gubler, U., Hellmann, C. P., Dukovich, M., Giri, J. G., Pan, Y-C E., Collier, K., Semionow, R., Chua, A. O., and Mizel, S. B. (1984) Nature 3 1 2 , 458-462 7. Auron, P. E., Webb, A. C., Rosenwasser, L. J., Mucci, S. F., Rich,
P. L. Simon, W. F. Fenderson, and J. T. Laydon, manuscript in preparation.
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.
11179
A., Wolff, S. M., and Dinarello, C. A. (1984) Proc. Natl. Acad. Sci. U. S. A. 8 1 , 7907-7911 Cameron, P., Limjuco, G., Rodkey, J., Bennett, C., and Schmidt, J. A. (1985) J. Exp. Med. 1 6 2 , 790-801 Kronheim, S. R., March, C. J., Erb, S. K., Conlon, P. J., Mochizuki, D. Y., and Hopp, T. P. (1985) J , Exp. Med. 161,490-502 Cameron, P. M., Limjuco, G . A., Chin, J., Silberstein, L. and Schmidt, J. A. (1986) J. Exp. Med. 1 6 4 , 237-250 Gubler, U.,Chua, A. O., Stern, A. S., Hellmann, C. P., Vitek, M. P., Dechiara, T. M., Benjamin, W. R., Collier, K. J., Dukovich, M., Familletti, P. C., Fiedler-Nagy, C., Jenson, J., Kaffka, K., Kilian, P. L., Stremlo, D., Wittreich, B. H., Woehle, D., Mizel, S. B., and Lomedico, P. T. (1986) J. Zmmunol. 136,2492-2497 Johanson, K., Miles, L., McDevitt, P., Chen, M-J., Holskin, B., Lillquist. J.. Young. P.. Simon.. P... and Mevers. C. (1986). Fed. Proc. 4 6 , 1719 (Ai;$tr.’1388) McCandliss. R.. Sloma., A.., and Pestka. S. (1981) Methods Enzymol. 79; 51-59 Maniatis. T.. Fritach. E. F.. and Sambrook. J. (1982) Molecular CloniG, a h r a t o k Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York Aviv, H., and Leder, P. (1972) Proc. Natl. Acad. Sci. U. S. A. 69, 1408-1412 Hanahan, D. (1983) J. Mol. Biol. 1 6 6 , 557-580 Beaucage, S. L., and Caruthers, M. H. (1981) Tetrahedron Lett. 22,1859-1862 McBride, L. J., and Caruthers, M. H. (1983) Tetrahedron Lett. 24,245-248 Gross, M., Sweet, R. W., Satbe, G., Yokoyama, S., Fasano, O., Goldfarb, M., Wigler, M., and Rosenberg, M. (1985) Mol. Cell. Biol. 5,1015-1024 Laemmli U. K. (1970) Nature 227,680-685 Simon, P. L., and Lee, J. C. (1986) J. Zmmunol. 1 3 7 , 557-562 Ellman, G. L. (1959) Arch. Biochem. Biophys. 8 2 , 70-77 Chane. C. T.., Wu., C-S. C.. and Yane. J. T. (1978) . . Anal. Biochem. m,-i3-31 Simon. P.. Lavdon. J. T.. and Lee. J. C. (1985) . , J. Immunol. M e t k s ‘ 84,“85-94 Van Damme, J., DeLey,M., Opdenakker, G., Billiau, A.,De Sommer, P., and Van Beeumen, J. (1985) Nature 3 1 4 , 266268 Simon, P. L., and Willoughby, W. F. (1981) J. Zmmunol. 126, 1534-1541 Edelhoch, H. (1967) Biochemistry 6,1948-1954 Johanson, K. O., Haynes, T. E., and McHenry, C. S. (1986) J . Biol. Chem. 2 6 1 , 11460-11465 Staehelin, T., Hobbs, D. S., Knug, H., Lai, C-Y., and Pestka, S. (1981) J. Biol. Chem. 2 6 6 , 9750-9754 Arakawa, T., Alton, N. K., and Hsu, Y-R. (1985) J. Biol. Chem. 260,14435-14439 Alson, K. C.,Fenno, J., Lin, N., Harkins, R. N., Snider, C., Kohr, W. H., Ross, M. J., Fodge, D., Prender, G., and Stebbing, N. (1981) Nature 293,408-411 Yamada, T., Kato, K., Kawahara, K., and Nishimura, 0. (1986) Biochem. Bwphys. Res. Commun. 135,837-843 Kronheim, S. R., Cantrell, M.A., Deeley, M.C., March, C. J., Glackin, P. J., Anderson, D.M., Hemenway, T., Merriam, J. E., Cosman, D., and Hopp, T. P. (1986) Biotechnology 4,10781082 Bradford, M. M. (1976) Anal. Biochern. 72,248-254 I
I
.
.
I
Continued on next page.
Human Recombinant Interleukin-1B
11180 SUPPLEHENTARY HATLRIAL TO PURIFICATION AN0CHARACTERIZATION OF HUMAN RECCWINANT INTERLEUKIN 18
-
€ w L w t r a t i o n and b u f f e r e x c h a n a The above p w l -as a d j u s t e d t o pH 5.0 and a p p l l e d t o a column (1.5x6cm) of S-Sepharore e q u i l i b r a t e d w l t h 2 5 M N H ~ M C .pH 5.2. A f t e r a 3-column volume wash. rIl-l8 was e l u t e dw i t h 0 . W N H ~ M c .pH 6.0. Up t o 30nglnl p r a t e l n Concentration has been obtalned.
Chester A. Heyerr. Kyung 0. Johanran. Lynette H. H l l e i . P a t r l c k J . HCOevltt. P h i l l p L. Slmn. R . Lee Webb. Yam-Jy Chen. Beverly P. H s l r k i n . Jay S . l l l l q u i s t . and Peter R . Young
-
-
h w n n w n ~ ~ t e rMonocytes were p r e p a w d from IO U n i t s o f p r m r a t l o n o f &vat$$ peripheralbloodobtalnadfromthe Red Cmsr v i ar e q u e n t l a lS e p a r a t l O n On FICOII and PITCOII g r a d i e n t s .f o l l o w e db ya t t a c h m e n tt op l a s t l cd i s h e sf o r 1.5h I n thepresence O f 11 humanA8 sorum. A f t e r remval o f "on-adherent c e l l s . t h em n a c y t e r rere theninduced t o d l f f e r e n t i a t e f o r 4h w i t h 300 n g h l LPS ( 7 ) .
c e l l % bytheguanidlnium CmstiULfiOnpf fONA l i b r a r y - T o t a l R(IA was preparedfromlysed Enrichmentforpoly A+ RNA lls t h i o C y l n l t e l m l U mc h l o r i d ec u s h i o n method (13.141. achlevedby o l i g o d l C h r m l t o g r a p h y ( 1 5 ) . Standard methods were used t o C o n l t w c t t h e LOHA l i b r a r y (14). except t h a t cloned HHLV l e v e r r e t r a n r c r l p t a r e was used t o synthesize the f l r l t s t r a n d cONA a c t o r d l n g t o t h e w p p l l e r $ ' r e t m e n d a t i o n r , and t h l s cONA was used w i t h o u t purificationbyadjurtlngthereactionmixturetothepropersaltconcentratlonfor second s t r a n dr y n t h e r l rf o l l o w e d by a d d i t i o n o f Klenew enzyme t o I n i t i a t e t h e reaction. Oliga dT Of pER322 v i a GC primed LDNAI O f m r e than 5W bp i n l e n g t h were I n s e r t e d I n t o t h e P s t I s i t e t a i l i n g . About 30.000 independent clones rere obtained upon h i g he f f i c i e n c yt r a n r f o r m a t i a n o f W294(161.
€=lm&al Seonencln% r I L - I 0 ( 3 Q 9 , 1 1 . 5 n m l ) "as incubated r l t h c a r b w p e p t l d a s e 7 ( 6 ~ 9 .0 . l n m l ) a t r w m temperature I n 1 2 q r l 0.5W w d l m acetate. pH 5.5 and 8H urea. DYPllcatP a l l q u o t l l5pl) were r e m v e d a t each t l m ep o l n t( F i 9 . 5 ) and frozen on d r y Ice f a r s t o r a g eU n t l lt h e yc o u l d be analyred. Each sample 11s dansylated r l t h a 12-fold excess (MI*) O f d a n r y lc h l o r i d ef a r 10 mln a t 650C I n 4 O d LI to A f t e r d l l Y t l o n r l t h 25nH sodium acetate. pH 6.5 ( 8 u f f e r A) each sample was lrne&?.?~ly loaded o n t o a Beckman Ultrasphere M)S Column (250 x 4 . 6 m )e q u l l l b r a t e d I n b u f f e r A made 51 w i t h CHjCN ( 8 u f f e r 8). Separation Of t h e d a n r y l - a d n o a d d s was a c c m p l i r h e db y increarlng t h ea m u n t O f buffer 8 i n bufferA 13 f a l l o r r : 301 8 i n A over 25min; 102 8 I n A W e ? thenext 7nln. Flow l a t e )la5 lmllmln. ONS-amino a d d s were I d e n t i f i e d b y f l u o r e s c e n t detection on a RcPherron m d e l Fl-749 s p e c t r o f l u o r m e t e r and q u a n t i t a t e d wing a S p e c t r a P h y l l C I m d e l SP4210 Computing I n t e g r a t o r .
-
s v n m e s i r and l a b l l i n o gf o l l ~ o n u c l e o t i d ee r n & Two oligonucleotides. derived from the published sequence O f human IL-10 and complementary a t t h e i r 3' ends, were synthesized u r l n g thesolid-phasephorphoramidlte method (17.18) on an automated A p p l i e d 8 i o l y l t e m ~ model 380A. Cyanoethylpharphoramiditer were used in p l a c e Of d i i l D p r D p y l p h o s p h o l l ~ i d i t e l . The o l l g o n u c l e a t i d e r had t h ef o l l o w i n g sequences: 5'-GTAATGACAIUUTACCTGTGGCCTT~~ClC-3' and 5'-GA~GGTACIGITTCTTTTCCTTGAGGCCCA-3'. For a l l g a n u c l e o t i d e Ilbelling. 1 m i x t u r e o f 0.1 pg of each CnplenentarYstrand was prlmerextendedwith 200 unl I of reverse t r a n s c r i p t a s e ( 3 7 9 , I h ) I n 5a* tdl. pH 8.1, c o n t a i n i n g 250vCi Of t h r e e3 i P - l a b e l l e d dNTPI and 5*n O f t h er e m a i n i n gu n l a b e l l e d dNTP. The f s l l - l e n g t hl a b e l l e d 50merr were separated from u n l a b e l l e d 361err by IS1 p r e p a r a t i v e PAGE. e l e c t r o e l u t e d . and used d i r e c t l y a f t e r bolllngforhybridlzatlont0filteri.
-
r nina f cONA I F i l t e r s were p r e h y b r i d l z e od v e r n i g hat t 42Oc i n Sxssc, 501 %r%de.'5x O e n h ~ d ~ o l u t l o n50nH . SodiUm phosphate ( pH 6.5). and lOOvg/ml denltured salmn sperm DNA. H y b r i d i z a t i o nw i t h 32P-labe11ed OligOnUcleotideprobes was done in the r a m r a l u t l o n exceptwith 21 Denhardt'ssolution. The f i l t e r s were e x t e n s i v e l yr a r h e d i n 2 x 8 s ~ a troom temperature plior t o autoradiography.
€!KLLU D i C h r p l U - CD spectra were obtained w i t h a Jasco m d e l J500C r p e c t r o p o l a r l n e t e r I n t e r f a c e d t o an I8H X L computer. Each f l n a l spectrum was an average of 8 s p e c t r a l scans w l t hw b t n c t i o n lo fs o l v e n tb l a n k .A n a l y s i s Of secandarystructure was a c c m p l l r h e d e s s e n t i a l l y by t h e method o f Chang e t . & I . ( 2 3 ) . r I L - I O was scanned a t 0.1-1 qlml i n t h e 5nU $odiumphMPhate. pH 6.5; lord4 Tris. pH 8 . 0 ; 5 M ammnium f o l l o u l n gb u f f e r r : acetate, pH 6.0.
-
81010~101 A m y The a b l l l t y Of I L - I 0 t o s t i m u l a t e t h e p r o d u c t i o n O f I n t e r l e u k i n 2 (IL-2) i n a culturedEl-4 murine T-cell l i n e 1 1 dose-dependent and can be measured by following t h e I n c o r p o r a t i o n O f t r i t i a t e d t h y m i d i n e i n t o an It-2-dependent mure c y t o t o x i c T-cell l l n e (241. The a l m y was performedexactly as described p r e v l o ~ ~ (l 2y4 ) .
RESULTSOF
Table 11. Amino AcldCamDoritionof Amino Acid
sequenceAverage
16.2
5.5
14
23 8
Pyrif~catlnn of rIL-IO:
8 5 2
2.0 10.5 5.9
11 6
4.1
5 15
4.2
4
9.1 14.9
9 I 15
NO 3.0
1 3
1 .I
-
17 6
23.1
15.0
low t r l r pH 1.5, 1 1 deoxycholate I1 T r i t o n X-IO0 0.11 50s). The f i l t e r 11s then exposed t o Kodak WR-5 film f o r 5 days 1; the prelence Of'a DupontKronex i n t e n s i f y i n g screen I t - 1 W C .
Predicted from (5.7) 1Reriduerla
11.1 8.2 7.8 4.1
VIL-IR
Human rlt-10.
R~ri4ws/wle
Pi a r a t i u e p l e r t i w h o r s i r and & L l i e e l u t i o n of rll-UL - i l l - 1 0 was expiessed as described ab%e fromhost AR13 a t4 1 scale. Sample l o a d i n gb u f f e r was added t ot h e cell l y r a t e $ u p e r n a t a n t p r i o r t o b o l l i n g and a p p l y i n g t o a p r e p a r a t i v e 2 0 1 805-polyacrylamide gel f o r e l e c t r o p h o r e l l l 120). A f t e r b r i e f Coomlslie blue v l r u a l l r a t i o n o f t h e rI1-10. t h eb m d war destained. Cut Out. and e l e c t r o e l u t e d o v e r n l g h t i n t o 0.05H NH4HCO3 c o n t a i n i n g 0.12 SOS. Excess g l y c i n e was r e m w d by e l e c t r o d l a l y i i r p r i o r t o I t T U c t U m I analyrir. The p r o t e i n * P I then I v m h i l i z e d . r e r u r ~ a n d e d i n w a t e r . and P r e c i p i t a t e dw i t he t h a n o lt o remVe SO2 and any
OF
PURIFICATION AN0CHARACTERIZATION
~~
* D e t e r m i n e db yp e r f o r m i ca c i dw i d a t i o n NO Not determlnod.
3 4 5
6 1 8
9 10 11 12
13 14
15 16 17 18 19 20
and x l d h y d r o l y i i r .
RecombinantHuman
Interleukin-la
11181
I
[ ,300
~"-""*
/='
40-
_/--
/--
100
200
Fractions
/.
/=
I
0
lo-
I
FIg.6
-
s"*
"
c
1
C - t e n l n a l sequence analyslr or rlL-18. TIme cwrsc release or amlna aclds bythe actlo" O f carboxypeptidase I I S S h m . The p r t d l c t r d IIqUeIICC a t the C-tenlnus of IL-I8 IS: ...Thr-Wct-Gln-Phe-val-Ser-Se~-~.
2000
4
loo0 0
c n
Flg.4
-
E -2000 Gel Flltratlon Chrmatograohy of rlL-18. The e l u t l o n p r o f l l c a t 28Onm 1s s h m . The show the result of SM-PAGE or altquots ( 1 % ~ ) rrm selected c01u.n fractlons. Gel electroDhoresls was run as described I n thelegend t o Flg.5. except ulthoutthe T U p r o t e l n p r e t l p l t a t l o n step. The peak was w l e d frm rractlons 55-62.
rn
190
30 20.1 67 43
14.4
200 250 240 230 220 210 rardength rm
Mr x 10.' 94-
-
Flg.7
-
