Refolding a glutamine synthetase truncation mutant in vitro: identifying superior conditions using a combination of...

A new method that uses a combination of bacterial GroE chaperonins and cellular osmolytes for in vitro protein folding is described. With this method, one can form stable chaperonin-protein folding intermediate complexes to prevent deleterious protein aggregation and, using these complexes, screen a large array of osmolyte solutions to rapidly identify the superior folding conditions. As a test substrate, we used GSDelta468, a truncation mutant of bacterial glutamine synthetase (GS) that cannot be refolded to significant yields in vitro with either chaperones or osmolytes alone. When our chaperonin/osmolyte method was employed to identify and optimize GSDelta468 refolding conditions, 67% of enzyme activity was recovered, comparable with refolding yields of wild type GS. This method can potentially be applied to the refolding of a broad spectrum of proteins.