Effect of chemical chaperones in improving the solubility of recombinant proteins in Escherichia coli

Department of Biotechnology, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar, Punjab 160 062, India.
Applied and Environmental Microbiology (Impact Factor: 3.95). 07/2011; 77(13):4603-9. DOI: 10.1128/AEM.05259-11
Source: PubMed

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.

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Available from: Shivcharan Prasad, Feb 07, 2014
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    • "Chemical chaperones are known to help correct the conformation of proteins via different mechanisms. Improved solubility has been observed for the cytokinin biosynthetic enzyme dimethylallyl pyrophosphate: 5 0 -AMP transferase (DMAPP: AMP transferase ), green fluorescent protein (GFP) and single-chain Fv when sorbitol was added to the growth medium [26] [27] [28]. Moreover, the addition of 0.4% glycerol to the culture medium enhanced the solubility of a human phenylalanine hydroxylase mutant enzyme [36], resulting in both higher solubility and activity. "
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    ABSTRACT: Human liver carboxylesterase 1 (CES1) plays a critical role in the hydrolysis of various ester-and amide-containing molecules, including active metabolites, drugs and prodrugs. However, it has been problematic to express recombinant CES1 in bacterial expression systems due to low solubility, with the CES1 protein being mainly expressed in inclusion bodies, accompanied by insufficient purity issues. In this study, we report an efficient in vitro method for refolding recombinant CES1 from inclusion bodies. A one-step purification with an immobilized-metal affinity column was utilized to purify His-tagged recombinant CES1. Conveniently, both denaturant and imidazole can be removed while the enzyme is refolded via buffer exchange, a dilution method. We show that the refolding of recombinant CES1 was successful in Tris–HCl at pH 7.5 containing a combination of 1% glycerol and 2 mM b-mercaptoethanol, whereas a mixture of other additives (trehalose, sorbitol and sucrose) and b-mercaptoethanol failed to recover a functional protein. His-tagged recombinant CES1 retains its biological activity after refolding and can be used directly without removing the fusion tag. Altogether, our results provide an alternative method for obtaining a substantial amount of functionally active protein, which is advantageous for further investigations such as structural and functional studies.
    Protein Expression and Purification 11/2014; 107:68-75. DOI:10.1016/j.pep.2014.11.006 · 1.51 Impact Factor
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    • "Improvement of recombinant protein solubility has been intensively studied for the purpose of overproducing soluble proteins, and various effective strategies have been reported, such as lowering incubation temperature [25], use of fusion partner [26], overexpression of chaperone proteins [27] and protein mutagenesis [28]. Recently, Prasad et al. reported a simple yet effective approach to increase the solubility of recombinant proteins, where sorbitol at high concentration reduced protein aggregation in E. coli[29]. To test if this approach could increase the solubility of DXS, high concentration of sorbitol was added directly to the cells in culture. "
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    ABSTRACT: Background: Recombinant proteins are routinely overexpressed in metabolic engineering. It is well known that some over-expressed heterologous recombinant enzymes are insoluble with little or no enzymatic activity. This study examined the solubility of over-expressed homologous enzymes of the deoxyxylulose phosphate pathway (DXP) and the impact of inclusion body formation on metabolic engineering of microbes. Results: Four enzymes of this pathway (DXS, ISPG, ISPH and ISPA), but not all, were highly insoluble, regardless of the expression systems used. Insoluble dxs (the committed enzyme of DXP pathway) was found to be inactive. Expressions of fusion tags did not significantly improve the solubility of dxs. However, hypertonic media containing sorbitol, an osmolyte, successfully doubled the solubility of dxs, with the concomitant improvement in microbial production of the metabolite, DXP. Similarly, sorbitol significantly improved the production of soluble and functional ERG12, the committed enzyme in the mevalonate pathway. Conclusion: This study demonstrated the unanticipated findings that some over-expressed homologous enzymes of the DXP pathway were highly insoluble, forming inclusion bodies, which affected metabolite formation. Sorbitol was found to increase both the solubility and function of some of these over-expressed enzymes, a strategy to increase the production of secondary metabolites.
    Microbial Cell Factories 11/2012; 11(1):148. DOI:10.1186/1475-2859-11-148 · 4.25 Impact Factor
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    ABSTRACT: The ascomycete Ophiostoma piceae produces a sterol esterase (OPE) with high affinity toward p-nitrophenol, glycerol, and sterol esters. Recently, this enzyme has been heterologously expressed in the methylotrophic yeast Pichia pastoris under the AOX1 methanol-inducible promoter (PAOX1) using sorbitol as co-susbtrate, and the hydrolytic activity of the recombinant protein (OPE*) turned out to be improved from a kinetic point of view. In this study, we analyze the effects of sorbitol during the expression of OPE*, at first added as an additional carbon source, and methanol as inducer. The O. piceae enzyme was successfully used for PVAc hydrolysis, suggesting its potential applicability in recycled paper production to decrease stickies problems.
    11/2012; 4(4). DOI:10.4161/bioe.22818
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