In vitro folding of human lyspro-proinsulin: Pathways and kinetics

Shuang Chen, Purdue University

Abstract

The in vitro folding of an oxidized proinsulin (methionine-arginine human lyspro-proinsulin S-sulfonate) was investigated, using cysteine as a reducing agent at 5°C and high pH (9.8-11.8). Folding intermediates were detected and characterized by means of matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), reversed-phase chromatography (RPC), size-exclusion chromatography (SEC), and gel electrophoresis (SDS-PAGE). The folding kinetics and yield depended on the protein and cysteine concentrations. RPC coupled with MALDI-MS analyses indicated a sequential formation of intermediates with one, two, and three disulfide bonds. The MALDI-MS analysis of Glu-C digested, purified intermediates indicated that an intra-A-chain disulfide bond formed first among A6, A7, and A11. Various non-native intra-A (A20 with A6, A7, or A11), intra-B (between B7 and B19), and inter-A-B disulfide bonds were observed in the intermediates with two disulfide bonds. The intermediates with three disulfide bonds had mainly the non-native intra-A and intra-B bonds. At a cysteine-to-proinsulin-SH ratio of 3.5, all intermediates with the non-native disulfide bonds were converted to properly folded proinsulin via disulfide bond reshuffling. Aggregation via the formation of intermolecular disulfide bonds of early intermediates was the major cause of yield loss. At a higher cysteine-to-proinsulin-SH ratio, some intermediates and folded MR-KPB-hPI were reduced to proteins with thiolate anions, which caused unfolding and even more yield loss than what resulted from aggregation of the early intermediates. Redox type, redox concentration, initial protein concentration, and folding pH were identified as important factors controlling folding kinetics and yield. Reducing protein concentration, while keeping an optimal redox-to-protein ratio, can improve folding yield significantly. A comprehensive kinetic model was developed to describe the disulfide-formation coupled folding of human lyspro-proinsulin. 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 was based on a reaction network which included folding initiated from both reduced and oxidized forms of proinsulin. The disulfide formation pathways of human lyspro-proinsulin via its sulfitolyzed (oxidized) form elucidated in this study provided strong experimental evidence for the proposed reaction network. The folding kinetic behaviors were investigated as a function of initial protein concentrations, pH, and redox conditions. Analyzing folding kinetics under several redox concentrations allowed a systematic estimation of kinetic constants. The estimated kinetic constants were sufficiently accurate to predict folding kinetics under a wide range of protein and redox concentrations. The model suggested that protein concentration, redox type, ratio, and concentration, and folding pH can be optimized to increase folding yield in vitro.

Degree

Ph.D.

Advisors

Wang, Purdue University.

Subject Area

Biochemistry|Chemical engineering

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