The conformational quality of insoluble recombinant proteins is enhanced at low growth temperatures

Abstract

Protein aggregation is a major bottleneck during the bacterial production of recombinant proteins. In general, the induction of gene expression at sub-optimal growth temperatures improves the solubility of aggregation-prone polypeptides and minimizes inclusion body (IB) formation. However, the effect of low temperatures on the quality of the recombinant protein, especially within the insoluble cell fraction, has been hardly ever explored. In this work, we have examined the conformational status of a recombinant GFP protein when produced in Escherichia coli below 37 degrees C. As expected, the fraction of aggregated protein largely decreased at lower temperatures, while the conformational quality of both soluble and aggregated GFP, as reflected by its specific fluorescence emission, progressively improved. This observation indicates that physicochemical conditions governing protein folding affect concurrently the quality of the soluble and the aggregated forms of a misfolding-prone protein, and that protein misfolding and aggregation are clearly not coincident events.

Hv-CBF2A overexpression in barley accelerates COR gene transcript accumulation and acquisition of freezing tolerance during cold acclimation

“…Such experimentation may be possible using a robust anti-CBF2 antibody that recognizes epitopes specific to CBF2 or an epitope-tagged recombinant CBF2, and chromatin immunoprecipitation. If the activity of the CBF2 protein in the plant is not altered by cold temperatures, the increase in the activity of the E. coliproduced recombinant proteins detected in vitro (Xue 2003;Skinner et al 2005) could be due to increased solubility, reduced misfolding, and reduced aggregation, all of which occur because the hydrophobic interactions contributing to these phenomena are weakened at colder temperatures (Baneyx and Mujacic 2004;Vera et al 2007).…”

Catalytically important amino-acid residues of abalone alginate lyase HdAly assessed by site-directed mutagenesis

“…In the present study, we could obtain sufficient amounts of soluble recHdAly for biochemical analysis using the cold-inducible yeast expression system. It was reported that solubility of proteins expressed in E. coli was considerably improved at low growth temperatures [36,37]. However, we have not succeeded yet to obtain sufficient amount of recombinant…”

Expression and Functional Characterization of the First Bacteriophage-Encoded Chaperonin

“…The bacterial expression of gp146 (61.7 kDa) was carried out at a lowered temperature (25°C) to increase the yield of soluble protein. Low-temperature induction is known to result in a significant improvement in solubility by slowing down the rate of protein expression and allowing time for folding (43). The recombinant protein was precipitated from the cell lysate by adding ammonium sulfate, followed by purification on a Q-Sepharose column (Fig.…”