Protein Misfolding and Inclusion Body Formation in Recombinant Escherichia coli Cells Overexpressing Heat-shock Proteins

Abstract

PreS2-S-␤-galactosidase, a three-domain fusion protein that aggregates extensively in the cytoplasm of Escherichia coli, was used to systematically investigate the effects of heat-shock protein (hsp) overproduction on protein misfolding and inclusion body formation. While the co-overexpression of the DnaK and DnaJ molecular chaperones led to a 3-6-fold increase in the recovery of enzymatically active preS2-S-␤-galactosidase over a wide range of growth temperatures (30 -42°C), an increase in the concentration of the GroEL and GroES chaperonins had a significant effect at 30°C only. Coimmunoprecipitation experiments confirmed that preS2-S-␤-galactosidase formed a stable complex with DnaK, but not with GroEL, at 42°C. When the intracellular concentration of chromosomal heat-shock proteins was increased by overproduction of the heat-shock transcription factor 32 , or by addition of 3% ethanol (v/v) to the growth medium, a 2-3-fold higher recovery of active enzyme was observed at 30 and 42°C, but not at 37°C. The overexpression of all heat-shock proteins or specific chaperone operons did not significantly affect the synthesis rates or stability of preS2-S-␤-galactosidase and did not lead to the disaggregation of preformed inclusion bodies. Rather, the improvements in the recovery of soluble and active fusion protein resulted primarily from facilitated folding and assembly. Our findings suggest that titration of the DnaK-DnaJ early folding factors leads to the formation of preS2-S-␤-galactosidase inclusion bodies.

Monitoring protein stability and aggregationin vivoby real-time fluorescent labeling

“…Our results complement previous efforts to follow protein aggregation in vivo (26)(27)(28)(29)(30). However, studies that allow direct observation of aggregation without analysis of cell constituents after lysis are rare (31,32 (33) designed an in vivo protein folding assay using N-terminal fusions to GFP to identify proteins that did not aggregate, but did not directly observe aggregation events.…”

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

“…shock prior to induction, possibly due to chaperone induction (12). Adding ethanol to the growth media mimics heat-shock response in E. coli and enhances solubility of some recombinant proteins (13)(14)(15).…”

Effects of GroESL Coexpression on the Folding of Nicotinoprotein Formaldehyde Dismutase fromPseudomonas putidaF61

“…[9][10][11] The magnitude of this eŠect is variable, apparently depending on the individual target protein to be overexpressed; in this study, coexpression of groESL markedly attenuated inclusion body formation, increasing FDM levels in the cell-free extract to 80z of the total. This is theˆrst report of GroELS assisting a nicotinoprotein to assume its native structure, including its tightly bound coenzyme, in vivo.…”

“…[6][7][8] For instance, coexpression of molecular chaperones-a ubiquitous class of proteins that play an essential role in protein folding, especially in vivo-increases the proportion of some recombinant proteins found in their native folded structure. [9][10][11] In that regard, it was recently reported that coexpressed molecular chaperones prevent heterologous proteins expressed by E. coli clones from forming inclusion bodies. For example, the activity of Anacystis nidulans ribulose bisphosphate carboxylase was increased by the coexpression of chaperonin.…”