Article

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: 3.67). 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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    • "Attempts have also been conducted to increase the fraction of soluble protein and catalytic activity expressed in E. coli by additives like sorbitol (Prasad et al. 2011) at different concentrations (Table 2). Sorbitol was found to have negative influence on the rate of cell growth (as measured by A 600 ), which suppress the E. coli BL21 (DE3) cell growth. "

    Full-text · Dataset · Oct 2015
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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.
    Full-text · Article · Nov 2014 · Protein Expression and Purification
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    • "Attempts have also been conducted to increase the fraction of soluble protein and catalytic activity expressed in E. coli by additives like sorbitol (Prasad et al. 2011) at different concentrations (Table 2). Sorbitol was found to have negative influence on the rate of cell growth (as measured by A 600 ), which suppress the E. coli BL21 (DE3) cell growth. "
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    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 colisystems 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 Ni2+ -NTA agarosegel column. The enzyme exhibited a higher affinity and substrate specificity for α-naphthyl acetate (α-NA), with kcat/Km of 100 s−1 μM−1 for α-NA, and the value is 29.78 s−1 μM−1 for β-naphthyl acetate. TheVmaxandKmwere also determined to be 12.9μmol/min/mg protein and 13.4 μM using substrateα-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 Cu2+ and Mg2+ 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. pomonellain vitro. This is the first report of the characterization of CEs protein from C.pomonella.
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