MUTATION OF ARG55 56 TO LEU55/ALA56 IN INSULIN-LIKE GROWTH FACTOR-I RESULTS IN 2 FORMS DIFFERENT IN DISULFIDE STRUCTURE AND NATIVE CONFORMATION BUT SIMILAR UNDER REVERSE-PHASE CONDITIONS/

Folding of recombinant human insulin-like growth factor-I (IGF-I) resu lts in two distinct species as resolved by reversed-phase high-perform ance liquid chromatography (RP-HPLC). The earlier eluting peak (PI) ha s a nonnative disulfide structure, while the later eluting peak (PII) assumes the native disulfide structure. This folding problem causes a lower yield and requires expensive RP-HPLC separation. In contrast, IG F-II folds mainly into a single form with all three disulfide bonds co rrectly formed. Sequence comparison of the two molecules revealed that IGF-I has arginine at residues 55 and 56, while IGF-II has alanine an d leucine, respectively, at these positions. Two analogs of IGF-I, IGF -I (Ala55/Leu56) and IGF-I (Leu56), behave similarly to IGF-II upon re folding and RP-HPLC; that is, a single peak eluted from the RP-HPLC co lumn. However, when the peaks isolated by RP-HPLC were subjected to hy drophobic interaction chromatography, circular dichroism, and peptide mapping, they were found to be a mixture of PI and PII. It was then co ncluded that factors other than just these two residues contribute to correct folding of IGF-II and that the PI and PII of the above two IGF -I mutants assume different conformation at neutral pH but similar con formation under the RP-HPLC condition.