Enhanced in vitro refolding of insulin-like growth factor I using a solubilizing fusion partner.

Abstract

We have previously shown that human insulin-like growth factor I (IGF-I), fused to ZZ (two domains derived from staphylococcal protein A), can be refolded at relatively high concentrations, without the use of solubilizing agents [Samuelsson, E., Wadensten, H., Hartmanis, M., Moks, T., & Uhlén, M. (1991) Bio/Technology 9, 363-366]. Here we have studied this phenomenon in detail by characterizing the in vitro refolding of IGF-I, fused to one or two solubilizing Z domains and without a solubilizing fusion partner. The characterization included solubility studies of the reduced proteins and an evaluation of the aggregation occurring during the refolding process. The results suggest that the applied fusion protein strategy can be used to obtain a cis-acting chaperone-like effect during refolding in vitro. Fusion to one or two Z domains resulted in more than a 100-fold increase in the solubility of reduced IGF-I. In addition, the Z or ZZ fusion partners decrease multimerization of the IGF-I moieties during the renaturation. The fusion protein strategy may be an option to overcome the obstacles of insolubility and aggregation, frequently encountered when designing in vitro refolding processes.