Article: article from journal or magazin.
Native folding of aggregation-prone recombinant proteins in Escherichia coli by osmolytes, plasmid- or benzyl alcohol-overexpressed molecular chaperones.
Cell Stress and Chaperones
When massively expressed in bacteria, recombinant proteins often tend to misfold and accumulate as soluble and insoluble nonfunctional aggregates. A general strategy to improve the native folding of recombinant proteins is to increase the cellular concentration of viscous organic compounds, termed osmolytes, or of molecular chaperones that can prevent aggregation and can actively scavenge and convert aggregates into natively refoldable species. In this study, metal affinity purification (immobilized metal ion affinity chromatography [IMAC]), confirmed by resistance to trypsin digestion, was used to distinguish soluble aggregates from soluble nativelike proteins. Salt-induced accumulation of osmolytes during induced protein synthesis significantly improved IMAC yields of folding-recalcitrant proteins. Yet, the highest yields were obtained with cells coexpressing plasmid-encoded molecular chaperones DnaK-DnaJ-GrpE, ClpB, GroEL-GroES, and IbpA/B. Addition of the membrane fluidizer heat shock-inducer benzyl alcohol (BA) to the bacterial medium resulted in similar high yields as with plasmid-mediated chaperone coexpression. Our results suggest that simple BA-mediated induction of endogenous chaperones can substitute for the more demanding approach of chaperone coexpression. Combined strategies of osmolyte-induced native folding with heat-, BA-, or plasmid-induced chaperone coexpression can be thought to optimize yields of natively folded recombinant proteins in bacteria, for research and biotechnological purposes.
Animals, Benzyl Alcohol/pharmacology, Chaperonin 10/metabolism, Chaperonin 60/metabolism, Escherichia coli/drug effects, Escherichia coli/genetics, Escherichia coli Proteins/chemistry, Escherichia coli Proteins/genetics, Gene Expression Regulation, Bacterial, Molecular Chaperones/genetics, Molecular Chaperones/metabolism, Osmolar Concentration, Protein Conformation, Protein Folding, Recombinant Fusion Proteins/chemistry, Recombinant Fusion Proteins/genetics
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