Abstract
Recombinant human insulin-like growth factor I (IGF-I) is efficiently expressed and secreted fromSaccharomyces cerevisiae using a yeast α-factor leader to direct secretion. However, approximately 10–20% of the IGF-I was in a monomeric form, the remaining materials being disulfide-linked aggregates. When the purified material was subjected to reverse-phase high-performance liquid chromatography (rp-HPLC), it gave two doublet peaks, I and II. Upon reduction, doublet peaks I and II converged to one doublet peak. This suggests that peaks I and II result from different disulfide structures, and the doublet feature of each peak results from other causes. Different disulfide structures between peaks I and II were also suggested from the near UV circular dichroism of these proteins. Only the peak II was biologically active, indicating that peak II has the correct disulfide structure. Concanavalin A affinity chromatography of the purified peak II doublet showed binding of the subpeak with an earlier rp-HPLC retention time, indicating that it was glycosylated. Sequence analysis of tryptic peptides suggested that Thr29 was the site of glycosylation. Site-directed mutagenesis was used to convert Thr29 to Asn29. This substitution reduced, but did not eliminate IGF-I glycosylation, suggesting additional glycosylation sites. The site of carbohydrate addition was consistent with the model that O-glycosylations occur on hydroxyl amino acids near proline residues in β-turns.
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References
Aubert, J. P., Biserte, G., and Loucheux-Lefebvre, M. H. (1976).Arch. Biochim. Biophys. 175, 410–418.
Bitter, G. A., Chen, K. K., Banks, A. R., and Lai, P. H. (1984).Proc. Natl. Acad. Sci. USA 81, 5330–5334.
Bitter, G. A., Egan, K. M., Koski, R. A., Jones, M. O., Elliott, S.G., and Griffin, J. C. (1987).Methods Enzymol. 153, 516–544.
Blundel, T. L., Bedarkar, S., Rinderknecht, E., and Humbel, R. E. (1978).Proc. Natl. Acad. Sci. USA 75, 180–184.
Blundell, T. L., Bedarkar, S., and Humbel, R. E. (1983).Fed. Proc. 42, 2592–2597.
Boyer, H. W., and Roulland, D. D. (1969).J. Mol. Biol. 41, 459.
Burleigh, B. D., and Meng, H. (1986).Cell Culture X, 48–53.
Copeland, K. C., Underwood, L. E., and Van Wyk, J. J. (1980).J. Clin. Endocrinol. Med. 50, 690–697.
D'Ercole, A. J., Underwood, L. E., Van Wyk, J. J., Decedue, C. J., and Foushee, D. B. (1976). In:Growth Hormones and Related Peptides, Excerpta Medica, Amsterdam, pp. 190–201.
Emr, S. D., Schekman, R., Flessel, M. D., and Thorner, J. (1983).Proc. Natl. Acad. Sci. USA 80, 7080–7084.
Ernst, J. F., Mermod, J.-J., DeLamarter, J. F., Mattaliano, R. J., and Moonen, P. (1987).Bio/Technol. 5, 831–834.
Ettinger, M. J., and Timasheff, S. N. (1971).Biochemistry 10, 824–831.
Fiat, A. M., Jolles, J., Aubert, J. P., Loucheux-Lefebvre, M. H., and Jolles, P. (1980).Eur. J. Biochem. 111, 333–339.
Fieschko, J. C., Egan, K. M., Ritch, T., Koski, R. A., Jones, M., and Bitter, G. A. (1987).Biotechnol. Bioeng. 29, 1113–1121.
Froesch, E. R., Schmid, C., Schwander, J., and Zapf, J. (1985).Ann. Rev. Physiol. 47, 443–467.
Greenfield, N., and Fasman, G. D. (1969).Biochemistry 8, 4108–4116.
Ito, H., Fukuda, Y., Murata, K., and Kimura, A. (1983).J. Bacteriol. 153, 163–168.
Jensenius, J. C., Anderson, I., Hau, J., Crone, M., and Koch, C. (1981).J. Immunol. Methods 46, 63–68.
Jonas, H. A., Newman, J. D., and Harrison, L. C. (1986).Proc. Natl. Acad. Sci. USA 83, 4124–4128.
Julius, D., Brake, A., Blair, L., Kunisawa, R., and Thorner, J. (1984).Cell 37, 1075–1089.
Kunkel, T. A., Roberts, J. D., and Zakour, R. A. (1987).Methods Enzymol. 154, 367–382.
Lehle, L., and Bause, E. (1984).Biochim. Biophys. Acta 799, 246–251.
Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982).Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
Moks, T., Abrahmsen, L., Osterlof, B., Josephson, S., Ostling, M., Enfors, S.-O., Persson, I., Nilsson, B., and Uhlen, M. (1987).Bio/Technol. 5, 379–382.
Peters, M. A., Lau, E. P., Snitman, D. L., Van Wyk, J. J., Underwood, L. E., Russell, W. E., and Svoboda, M. E. (1985).Gene 35, 83–89.
Raschdorf, F., Dahinden, R., Maerki, W., and Richter, W. J. (1988).Biomed. Environmental Mass Spectrometry 16, 3–8.
Rinderknecht, E., and Humbel, R. E. (1978).J. Biol. Chem. 253, 2769–2776.
Schoenle, E., Zapf, J., Humbel, R. E., and Froesch, E. R. (1982).Nature 296, 252–253.
Skottnev, A., Clark, R. G., Robinson, I. C. A. F., and Fryklund, L. (1982).J. Endocrinol 112, 123–132.
Takayaso, T., Suzuki, S., Kametani, F., Takahashi, N., Shinoda, T., Okuyama, T., and Munekata, E. (1982).Biochem. Biophys. Res. Commun. 105, 1066–1071.
Tschumper, G., and Carbon, J. (1983).Gene 23, 221–232.
Viera, J., and Messing, J. (1978).Methods Enzymol. 153, 3–11.
Yanisch, Perron, C., Viera, J., and Messing, J. (1985).Gene 33, 103–119.
Zapf, J., Froesch, E. R., and Humbel, R. E. (1981).Curr. Top. Cell. Regul. 19, 257–309.
Zsebo, K. M., Lu, H.-S., Fieschko, J. C., Goldstein, L. Davis, J., Duker, K., Suggs, S. V., Lai, P.-H., and Bitter, G. A. (1986).J. Biol. Chem. 261, 5858–5865.
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Elliott, S., Fagin, K.D., Narhi, L.O. et al. Yeast-derived recombinant human insulin-like growth factor I: Production, purification, and structural characterization. J Protein Chem 9, 95–104 (1990). https://doi.org/10.1007/BF01024990
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DOI: https://doi.org/10.1007/BF01024990