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.Overexpression of proteins in heterologous systems often results in the formation of inclusion bodies. These electronrefractile particles are formed because of the failure of prokaryotic folding machinery to correctly fold the nascent polypeptide chain, increasing its local concentration in the cytosol. The most common strategy to recover active proteins from these particles is by denaturing them in the presence of chaotropes and refolding them to the native conformation by the gradual removal of the denaturant. This process results in high loss in protein yield since the renaturation efficiency of most proteins is not very high.

Influence of Osmotic Shock on <b><i>Escherichia coli</i></b> Insoluble Protein Fraction in the Presence of Exogenous Osmolytes

“…The same effect was also observed with glycine [Diamant et al, 2001[Diamant et al, , 2003]. In accordance with the experiments of Prasad et al [2011], osmolytes help in the solubilization of proteins if they can permeate the cell membrane and participate in ATP generation. In our case, betaine is not a compound metabolized by E. coli , but it has a positive effect on the solubility of IFN-α5.…”

“…Usually, the addition of osmolytes is combined with other methods such as osmotic and/or heat shock or plasmid-induced chaperone co-expression [Choi et al, 2010;De Marco et al, 2005;Oganesyan et al, 2007;Schultz et al, 2007]. In a recent paper, Prasad et al [2011] have attempted to understand the mechanism by which the osmolytes increase the solubility of heterologous protein. They found that the solubilization of green fluorescent protein in vivo was proportional to the concentration of ATP produced in the cells.…”

“…They found that the solubilization of green fluorescent protein in vivo was proportional to the concentration of ATP produced in the cells. They concluded that, despite the complexity of the mechanism, only those osmolytes which are able to permeate the cell membrane and which are potential participants in ATP generation pathways are solubilizers of overexpressed recombinant proteins in vivo [Prasad et al, 2011].…”

Enhanced Solubilization of Class B Radical S-Adenosylmethionine Methylases by Improved Cobalamin Uptake in Escherichia coli

“…For example, it is well known that arginine and other metabolites such as trehalose and glycine betaine, among others, can enhance in vivo protein folding. 68, 69 The enhancement in solubility might also be due to simple differences in metabolism between E. coli using ethanolamine as an energy source and E. coli using glucose.…”

Synergistic Inhibition of Protein Fibrillation by Proline and Sorbitol: Biophysical Investigations

“…Sorbitol is known to improve production of soluble recombinant proteins in E. coli [33] and also increases solubility of intracellular FVIII aggregates, hence improves transport from endoplasmic reticulum to golgi in mouse models [34]. Sorbitol is also reported to be involved in initiation and facilitation of proper folding of several mutants of human cystathionine β-synthase [35].…”