YEAST-DERIVED RECOMBINANT HUMAN INSULIN-LIKE GROWTH FACTOR-I - PRODUCTION, PURIFICATION, AND STRUCTURAL CHARACTERIZATION

被引:38
作者
ELLIOTT, S [1 ]
FAGIN, KD [1 ]
NARHI, LO [1 ]
MILLER, JA [1 ]
JONES, M [1 ]
KOSKI, R [1 ]
PETERS, M [1 ]
HSIEH, P [1 ]
SACHDEV, R [1 ]
ROSENFELD, RD [1 ]
ROHDE, MF [1 ]
ARAKAWA, T [1 ]
机构
[1] AMGEN INC,1900 OAK TERRACE LANE,THOUSAND OAKS,CA 91320
来源
JOURNAL OF PROTEIN CHEMISTRY | 1990年 / 9卷 / 01期
关键词
circular dichroism; disulfide pairing; glycosylation; Insulin-like growth factor; mutation;
D O I
10.1007/BF01024990
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Recombinant human insulin-like growth factor I (IGF-I) is efficiently expressed and secreted from Saccharomyces 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. © 1990 Plenum Publishing Corporation.
引用
收藏
页码:95 / 104
页数:10
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