Abstract: | The crucial step of folding of recombinant proteins presents serious challenges to obtaining the native structure. This problem is exemplified by insulin‐like growth factor (IGF)‐I which when refolded in vitro produces the native three‐disulfide structure, an alternative structure with mispaired disulfide bonds and other isomeric forms. To investigate this phenomenon we have examined the refolding properties of an analog of IGF‐I which contains a 13‐amino acid N‐terminal extension and a charge mutation at position 3 (Long‐ [Arg3]IGF‐I). Unlike IGF‐I, which yields 45% of the native structure and 24% of the alternative structure when refolded in vitro, Long‐[Arg3]GF‐I yields 85% and 10% of these respective forms. To investigate the interactions that affect the refolding of Long‐[Arg3]IGF‐I and IGF‐I, we acid‐trapped folding intermediates and products for inclusion in a kinetic analysis of refolding. In addition to non‐native intermediates, three native‐like intermediates were identified, that appear to have a major role in the in vitro refolding pathway of Long‐[Arg3]IGF‐I; a single‐disulfide Cys18–Cys61 intermediate, an intermediate with Cys18–Cys61 and Cys6–Cys48 disulfide bonds and another with Cys18–Cys61 and Cys47–Cys52 disulfide bonds. Furthermore, from our kinetic analysis we propose that the Cys18‐Cys61, Cys6‐Cys48 intermediate forms the native structure, not by the direct formation of the last (Cys47‐Cys52) disulfide bond, but by rearrangement via the Cys18–Cys61 intermediate and a productive Cys18–Cys61, Cys47–Cys52 intermediate. In this pathway, the last disulfide bond to form involves Cys6 and Cys48. Finally, we apply this pathway to IGF‐I and conclude that the divergence in the in vitro folding pathway of IGF‐I is caused by non‐native interactions involving Glu3 that stabilize the alternative structure. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 693–703, 1999. |