Improving the invitr0 folding of disulfide containing protein

Yingsong Wang, Shweta Mandumula, and Watson J. Lees

All from the Department of Chemistry and Biochemistry, Florida International University

Oxidative protein folding efficiency is critical as many recombinant proteins contain disulfide bonds and need to be folded in vitro after forming inclusion bodies during overexpression in bacteria.^[1] However, in vitro protein folding can be hindered by the need to escape from kinetically trapped intermediates which accumulate during folding and only slowly react to form native protein. Historically, in vitro oxidative folding studies of bovine pancreatic trypsin inhibitor (BPTI) focused on characterizing the folding pathway but not on folding efficiency. Therefore, the folding pathway of BPTI has served as a paradigm for the folding of disulfide-containing proteins but folding took weeks at physiological pH because of the need to escape from kinetic traps via a rearrangement type pathway. By determining rate constant for the reactions of the kinetic traps, we demonstrate that growth type pathways, direct oxidation of the remaining thiols to native disulfides, are feasible and can be more efficient than rearrangement pathways. Folding via a growth type pathway using an oxidation, reduction and oxidation cycle dramatically improves BPTI folding efficiency and may become a general methodology for protein production.

Thursday or Saturday would be better as I teach Friday morning