Abstract:

Target proteins overexpressed in Escherichia coli often result in the formation of inclusion bodies, which need further refolding in vitro to gain their native structures and biological functions. In order to improve the refolding rate by dilution refolding method, recombinant green fluorescent protein (GFP) inclusion bodies were used as the model to develop a novel dual denaturation- dilution refolding method in this study. This novel method used the alkaline solution containing arginine as the first denaturant to dissolve GFP inclusion bodies, and then exhibited a gradient reduction in pH of the solution to mildly form the arget protein aggregates. The denatured aggregates were refolded by dilution in urea solution. Results indicated that the recovery rate of refolded GFP by this novel method was over 80%, which was 1.5 to 2.3 times higher than that of traditional methods. Moreover, the stability of refolded GFP by this novel method was highly consistent with the natural protein. Therefore, this developed method for protein refolding could be used to refold proteins, especially GFP-related proteins from inclusion bodies with high efficiency. This investigation will also provide optimal methods for protein refolding from inclusion bodies through combinatorial design and extended applications of different denaturants.

Key words: Escherichia coli, inclusion body, denaturation, refolding, green fluorescent protein