Effect of chemical chaperones in improving the solubility of recombinant proteins in Escherichia coli.
ABSTRACT The recovery of active proteins from inclusion bodies usually involves chaotrope-induced denaturation, followed by refolding of the unfolded protein. The efficiency of renaturation is low, leading to reduced yield of the final product. In this work, we report that recombinant proteins can be overexpressed in the soluble form in the host expression system by incorporating compatible solutes during protein expression. Green fluorescent protein (GFP), which was otherwise expressed as inclusion bodies, could be made to partition off into the soluble fraction when sorbitol and arginine, but not ethylene glycol, were present in the growth medium. Arginine and sorbitol increased the production of soluble protein, while ethylene glycol did not. Production of ATP increased in the presence of sorbitol and arginine, but not ethylene glycol. A control experiment with fructose addition indicated that protein solubilization was not due to a simple ATP increase. We have successfully reproduced these results with the N-terminal domain of HypF (HypF-N), a bacterial protein which forms inclusion bodies in Escherichia coli. Instead of forming inclusion bodies, HypF-N could be expressed as a soluble protein in the presence of sorbitol, arginine, and trehalose in the expression medium.
SourceAvailable from: Xueqing Yang[Show abstract] [Hide abstract]
ABSTRACT: Carboxylesterases (CEs) are enzymes responsible for the detoxification of insecticides in insects. In the Cydia pomonella, CEs are involved in synthetic pyrethroid, neonicotinoid, carbamate, and organophosphate detoxification. However, functional overexpression of CEs proteins in Escherichia coli systems often results in insoluble proteins. In this study, we expressed the fusion protein CpCE-1 in E. coli BL21 (DE3). This recombinant protein was overexpressed as inclusion bodies at 37 °C whereas it produced a higher percentage of soluble protein at lower growth temperatures. Production of soluble proteins and enzyme activity increased in the presence of sorbitol in the growth medium. The fusion protein was purified from the lysate supernatant using a Ni(2+)-NTA agarose gel column. The enzyme exhibited a higher affinity and substrate specificity for α-naphthyl acetate (α-NA), with k cat/K m of 100 s(-1) μM(-1) for α-NA, and the value is 29.78 s(-1) μM(-1) for β-naphthyl acetate. The V max and K m were also determined to be 12.9 μmol/min/mg protein and 13.4 μM using substrate α-NA. The optimum pH was 7.0 and temperature was 25 °C. An enzyme inhibition assay shows that PMSF and DEPC strongly inhibit the enzyme activity, while the metal ions Cu(2+) and Mg(2+) significantly activated the activity. More importantly, cypermethrin, methomyl, and acephate were found to suppress enzyme activity. The data demonstrated here provide information for heterologous expression of soluble protein and further study on insecticide metabolism in C. pomonella in vitro. This is the first report of the characterization of CEs protein from C. pomonella.Applied Microbiology and Biotechnology 09/2013; 97(24). DOI:10.1007/s00253-013-5236-8 · 3.81 Impact Factor
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ABSTRACT: Peptide dendrimers are screened for “artificial chaperone” (protein refolding) activity by a sensitive fluorescence based assay. The refolding with largest dendrimer is found to help in recovering biological activity of >90% in the case of unfolded lipases and amylases. The refolding yields decrease down to 14% with a decrease in the complexity and hydrophobicity of the dendron/dendrimer. CD spectroscopy confirms the correct refolding in terms of secondary structure contents of the proteins. The DLS data indicates that presence of the dendrons/dendrimers facilitates protein refolding by preventing the aggregation of proteins.RSC Advances 05/2013; 3(21):8016-8020. DOI:10.1039/C3RA00061C · 3.71 Impact Factor
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ABSTRACT: Biologically active proteins are useful for studying the biological functions of genes and for the development of therapeutic drugs and biomaterials in a biotechnology industry. Overexpression of recombinant proteins in bacteria, such as Escherichia coli, often results in the formation of inclusion bodies, which are protein aggregates with non-native conformations. As inclusion bodies contain relatively pure and intact proteins, protein refolding is an important process to obtain active recombinant proteins from inclusion bodies. However, conventional refolding methods, such as dialysis and dilution, are time consuming and, often, recovered yields of active proteins are low, and a trial-and-error process is required to achieve success. Recently, several approaches have been reported to refold these aggregated proteins into an active form. The strategies largely aim at reducing protein aggregation during the refolding procedure. This review focuses on protein refolding techniques using chemical additives and laminar flow in microfluidic chips for the efficient recovery of active proteins from inclusion bodies.03/2014; 4(1):235-51. DOI:10.3390/biom4010235