Yeast-derived recombinant human insulin-like growth factor I: production, purification, and structural characterization.
Recombinant human insulin-like growth factor I (IGF-I) is efficiently expressed and secreted from Saccharomyces cerevisiae using a yeast alpha-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 beta-turns.
Amgen Inc., Thousand Oaks, California 91320.
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S
Elliott
K D
KD
Fagin
L O
LO
Narhi
J A
JA
Miller
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M
Jones
R
R
Koski
M
M
Peters
P
P
Hsieh
R
R
Sachdev
R D
RD
Rosenfeld
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D006031:Glycosylation; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000032:analysis* / Q000096:biosynthesis / Q000648:ultrastructure; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000648:ultrastructure; D012441:Saccharomyces cerevisiae / Q000032:analysis*; D013002:Somatomedins / Q000032:analysis*
D011994:Recombinant Proteins; D013002:Somatomedins; D007334:Insulin-Like Growth Factor I