Native folding of aggregation-prone recombinant proteins in Escherichia coli by osmolytes, plasmid- or benzyl alcohol–overexpressed molecular chaperones

Abstract

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-DnaJGrpE, 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 plasmidinduced chaperone coexpression can be thought to optimize yields of natively folded recombinant proteins in bacteria, for research and biotechnological purposes.

Membrane lipid composition affects plant heat sensing and modulates Ca2+-dependent heat shock response

“…These findings are in line with earlier reports that membrane fluidizers, such as benzyl alcohol, cause a HSR at non-inducing temperatures in plants and bacteria. 11,12,28 Thus, in Escherichia coli 19 and Synechocystis 29 preadaptation to different growth temperatures modulated the subsequent heat-induced expression levels of major classes of HSPs and affected thermotolerance.…”

“…[8][9][10] Yet, the overexpression of HSPs, so called the heat shock response (HSR), can be activated under mild physiological conditions that are unlikely to cause any protein denaturation in the cell. [11][12][13] Although multiple HSR triggering mechanisms may co-exist, 6,14-16 recent evidences in bacteria, algae, plants and mammalian cells point at the understanding how plants sense and respond to heat stress is central to improve crop tolerance and productivity. recent findings in Physcomitrella patens demonstrated that the controlled passage of calcium ions across the plasma membrane regulates the heat shock response (hSr).…”

Effect of osmotic stress and heat shock in recombinant protein overexpression and crystallization

“…This results in enhancement of protein stability (19,20). It has been shown with several proteins that some osmolytes, when added to the growth media, assist protein folding resulting in increased protein solubility (21)(22)(23). Preadaptation of E. coli cells to increasingly high concentrations of salt in the media leads to crosstalk between osmolytes and heat-shock proteins and decreases the tendency of soluble proteins to form insoluble aggregates during heat-shock (24).…”

Hyperfluidization-coupled membrane microdomain reorganization is linked to activation of the heat shock response in a murine melanoma cell line

“…Because the raft structure is also dependent on the thermally controlled lipid-phase behavior, we assumed that even mild changes in temperature could result in a fundamentally altered solubility and consequently in the redistribution and activity of potential stress-sensing/signaling proteins in the rafts (4,5). We recently furnished evidence that the drug candidate bimoclomol, its analogs, and the widely used membrane fluidizer benzyl alcohol (BA) are all capable of activating cellular Hsp formation without causing measurable protein denaturation (9)(10)(11)(12)(13).…”