Development of an optimized refolding process for recombinant Ala–Glu–IGF-1

Denatured and reduced N-terminal extended insulin-like growth factor-1 (AE-IGF-1) was purified from Escherichia coli extracts and subjected to in vitro folding. The renaturation process was shown to be a function of the redox potential of the solution. Folding by different methods had no significant effect on the renaturation. A maximal yield of 60% (w/w)was obtained. The folded AE-IGF-1 was enzymatically converted to IGF-1. The major by-product (20% w/w) was identified as scrambled IGF-1. Enzymatic digestion at alkaline and acidic pH suggested two possible disulphide bond arrangements: (i) Cys6–Cys47, Cys18–Cys61, Cys48–Cys52; or (if) Cys6–Cys52, Cysl8–Cys61, Cys47 and Cys48 being in their reduced forms. Energy minimization and molecular modelling suggested that the scrambled IGF-1, having reduced cysteines at positions 47 and 48, was the energetically most stable conformation of the two.