Increasing yield in the in vitro folding of Methionine-Arginine Human Lyspro Proinsulin-S-Sulfonate
Abstract
The refolding of MR-KPB-hPSS at alkaline pH using cysteine as the reducing agent was investigated. Aggregates of proinsulin-S-SO 3- and folding intermediates were characterized with Fluorescence Correlation Spectroscopy, Size Exclusion Chromatography, and Reversed-Phase HPLC. Flocculation of MR-KPB-hPSS was induced by 20-50% (v/v) IPA, high salt concentration, and low pH. The elimination of positive charges by dissolving hPSS in 0.1 N NaOH was found to reduce hPSS to a primarily monomeric form, however the dissolution of hPSS at these conditions resulted in misfolding. The folding yield of hPI was improved by reducing aggregation with the timely addition of a strong reducing agent. Through optimization of the dissolution and folding conditions, folding yields of up to 75% were obtained for 1 g/L solutions.
Degree
M.S.Ch.E.
Advisors
Wang, Purdue University.
Subject Area
Chemical engineering
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