Regulation of Protein Folding using Organic Solvents and Ionic Liquids

Abstract

The Escherichia coli expression system is frequently used to prepare various proteins in a highly efficient manner using information obtained by the Human Genome Project. However, recombinant proteins are often expressed as inclusion bodies which are biologically inactive, and a refolding reaction is absolutely required to form the correct tertiary structure. For this purpose, in vitro refolding reactions of recombinant proteins are generally performed in aqueous solutions. However, it is still difficult to efficiently fold recombinant proteins into their biologically active form in aqueous solutions because aggregates are formed as the result of the hydrophobic folding intermediates. Ionic liquids have recently been employed for refolding reaction of proteins in place of aqueous solutions and successfully folded into their native conformations can be accomplished, although the folding yield was still low. In addition, organic solvents, such as trifluoroethanol, are generally used for protein folding to induce α-helix formation and may reduce the extent of hydrophobic interactions. Therefore, to regulate or suppress hydrophobic interactions during the refolding reaction, organic solvents and ionic liquids were examined in attempts to construct the native conformation or to produce a proper folding intermediate.Dioxiane and 1-hexyl-3-methylimidazolium chloride (HMIM) were examined for the refolding reaction of prouroguanylin, as a model protein, which contains three disulfide bonds. Prouroguanylin was able to fold into its native conformation at a low concentration of HMIM or dioxane. Surprisingly, prouroguanylin was still able to form native disulfide bonds even at 80% HMIM or dioxane although prouroguanylin was not able to fold into its native structure under denaturing condition using urea or guanidine hydrochloride. Therefore, the results indicate that those solvents are suitable candidate solvents for use in protein folding studies. The results will be discussed in this paper.