Probing the disulfide folding pathway of insulin-like growth factor-I

The crucial step of folding of recombinant proteins presents serious challe nges to obtaining the native structure. This problem is exemplified by insu lin-like growth factor (IGF)-I which when refolded in vitro produces the na tive three-disulfide structure, an alternative structure with mispaired dis ulfide bonds and other isomeric forms. To investigate this phenomenon we ha ve 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 (Lo ng-[Arg(3)]IGF-I). Unlike IGF-I, which yields 45% of the native structure a nd 24% of the alternative structure when refolded in vitro, Long-[Arg(3)]IG F-I yields 85% and 10% of these respective forms. To investigate the intera ctions that affect the refolding of Long-[Arg(3)]IGF-I and IGF-I, we acid-t rapped folding intermediates and products for inclusion in a kinetic analys is 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-[Arg(3)]IGF-I; a single-disulfide Cys(18)-C ys(61) intermediate, an intermediate with Cys(18)-Cys(61) and Cys(6)-Cys(48 ) disulfide bonds and another with Cys(18)-Cys(61) and Cys(47)-Cys(52) disu lfide bonds. Furthermore, from our kinetic analysis we propose that the Cys (18)-Cys(61), Cys(6)-Cys(48) intermediate forms the native structure, not b y the direct formation of the last (Cys(47)-Cys(52)) disulfide bond, but by rearrangement via the Cys(18)-Cys(61) intermediate and a productive Cys(18 )-Cys(61), Cys(47)-Cys(52) intermediate. In this pathway, the last disulfid e bond to form involves Cys(6) and Cys(48). Finally, we apply this pathway to IGF-I and conclude that the divergence in the in vitro folding pathway o f IGF-I is caused by non-native interactions involving Glu(3) that stabiliz e the alternative structure. (C) 1999 John Wiley & Sons, Inc.