Optimization Of The Procedure For Protein Refolding From Inclusion Bodies And The Investigation Of Refolding Mechanism

The utilization of Escherichia coli expression system for foreign recombinant protein production is the most conventional strategy. However, the expressed recombinant proteins often accumulate in insoluble and inactive deposits called inclusion bodies. The unfolding of the inclusion body and the refolding of denatured protein to their native conformation with biological properties are the major bottlenecks.In this study, we use the technique developed by our group-two step denaturing and refolding process of inclusion bodies to optimize the denaturing and refolding procedure of EGFP (Enhanced Green Fluorescent Protein), MMP-12(Matrix Metal Proteinases-12 Catalytic Domain) and DBD (DNA Binding Domain) motif of NRSF/REST. During the unfolding process of the three proteins, firstly, we use 7M guanidine hydrochloride as the first denaturant to dissolve the inclusion body; second, we employ gradient dilution method to precipitate the target protein; third, we chose 8M urea as the second denaturant to dissolve the precipitation. Rapid dilution method was adopted to refold EGFP. As a result, the activity yield of EGFP obtained through two step-denaturing and refolding procedure is significantly 33.8% higher than one-step denaturing and refolding method. By using stepwise dialysis method for refolding technique, about 45 mg of MMP-12 was achieved from 1-liter of LB medium, exhibiting double folds of the amount (23mg) of MMP-12 obtained from the one step-denaturing and refolding technique, with comparable biological activity. Despite the failure to refold DBD back to its bioactive form through one-step denaturing and refolding method,7mg of refolded DBD could be gained utilizing the above two denaturants and gradient dialysis refolding method.Morever, in order to study the refolding mechanism of MMP-12, the refolding process was monitored by NMR, intrinsic tryptophan fluorescence, Circular dichroism spectroscopy and ANS fluorescence. The midpoint for the refolding of MMP-12 obtained from NMR coincides with those obtained from intrinsic tryptophan fluorescence, CD spectroscopy and ANS fluorescence (Cm≈4). From the HSQC spectrums of MMP-12 at different urea concentrations, we can learn that the signal gathers evidently when the urea concentration is higher than 4M, while the HSOC spectrum shows a nice dispersion in chemical shifts when the urea concentration is no more than 3M. From the above information we may conclude that the protein folds via a two-state mechanism without accumulating stable intermediates.