(677f) Kinetic Analysis of the in Vitro Folding of Methionine-Arginine Lyspro Proinsulin

The in vitro folding of sulfitolyzed (oxidized) human lyspro-proinsulin in the presence of cysteine was investigated in our previous study. The disulfide formation pathways were elucidated, and the structures of the intermediates were characterized. (1) The results showed that the folding yield varied strongly with pH, protein concentration, and redox concentrations. The yield was limited by the formation of aggregates and kinetically trapped intermediates. In this study, a comprehensive kinetic model was developed based on the pathways and the observed intermediates. The model described the folding process as a series of reversible inter- and intra-molecular thiol-disulfide exchange reactions with reversible covalent aggregation as the off-pathway reaction. In addition, the model included folding initiated from both reduced and oxidized forms of the protein. The temporal curves of the intermediates with one, two, and three disulfide bonds were obtained under a wide range of redox conditions and protein concentrations. The data allowed systematic estimation of the kinetic constants of the model. The model was used to find the folding conditions to reduce formation of the aggregates and to increase yield. The results were verified with experimental data. A similar method could be applied to optimize the folding yield of other disulfide containing proteins.

1.S Chen, L Adijanto, N.-H. L. Wang, ?In vitro folding of methionine-arginine human lyspro-proinsulin s-sulfonate - disulfide formation pathways and factors controlling yield?,2010 Biotechnology Progress (accepted, DOI: 10.1002/btpr.439)