Refolding a glutamine synthetase truncation mutant in vitro: identifying superior conditions using a combination of chaperonins and osmolytes.
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.
Voziyan
P A
PA
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, Kansas 66160-7421, USA.
Jadhav
L
L
Fisher
M T
MT
D018835:Chaperonin 10 / Q000494:pharmacology; D018834:Chaperonin 60 / Q000494:pharmacology; D018833:Chaperonins / Q000494:pharmacology*; D005974:Glutamate-Ammonia Ligase / Q000737:chemistry*; D005990:Glycerol / Q000494:pharmacology; D009154:Mutation; D017510:Protein Folding
D018835:Chaperonin 10; D018834:Chaperonin 60; D018833:Chaperonins; D005974:Glutamate-Ammonia Ligase; D005990:Glycerol