Improved renaturation process of aggregated recombinant proteins through the design of hydrated ionic liquids

Kyoko FUJITA¹, Kazune KOBAYASHI¹, Anna ITO¹, Shun YANAGISAWA¹, Kimiyoshi ICHIDA¹, Kota TAKEDA², Nobuhumi NAKAMURA³, Hiroyuki OHNO^3
1Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan
²The University of Tokyo, Tokyo, Japan
³Tokyo University of Agriculture and Technology, Tokyo, Japan

Recombinant expression using Escherichia coli (E. coli) as a host is the most commonly used technology to obtain target proteins. However, a concerning and unaddressed issue is that a considerable amount of inactive aggregation is obtained at a high rate, and despite several attempts, no simple, fast, and effective method for regenerating aggregated proteins in E. coli has been developed to date. More efficient and feasible method is needed to restore aggregated proteins expressed in E. coli. We have studied the potential of hydrated ILs as liquid chaperones, which induce the refolding behaviour of proteins, such as lyophilised, thermally aggregated, and aggregated proteins, during recombinant expression. However, in the case of aggregated protein, the solubility in hydrated ILs was low. In this study, we aimed to design and evaluate ILs capable of renaturation the activity of aggregated recombinant proteins, which involves a balance between high solubility and inducing the refolding of the dissolved protein.

CcCel6 Cellulase 6A from Coprinopsis cinerea (CcCel6A) was used as the target protein. For evaluation, the effects of ion structure, alkyl chain length of IL, and water content on the solubility and folding state of the aggregated recombinant protein were measured spectroscopically. Furthermore, the activity of the dissolved CcCel6A in hydrated ILs was measured after the transition of CcCel6A in buffer. Two transition methods were investigated as a final renaturation process to obtain highly active CcCel6A in buffer.

The ILs, as shown in Fig. 1, were selected to investigate the solubility of aggregated CcCel6A. Aggregated CcCel6A was mixed with hydrated ILs and tris buffer and stirred overnight. Most of the aggregated CcCel6A precipitate remained in the buffer solution; in contrast, a certain level of dissolution of the precipitation was observed in hydrated ILs by visual examination. Fluorescence spectra of the IL-treated solution, which is the supernatant obtained after centrifugation of aggregated CcCel6A mixed and stirred in hydrated IL were measured to compare the solubility. The fluorescence maxima differed depending on the alkyl chain length of the cation rather than the difference between the cation and anion type. ILs composed of tetrabutylammonium or tetrabutylphosphonium cations showed improved solubilization ability than those with different numbers of alkyl and butyl chains. Furthermore, the folding state of CcCel6A treated in hydrated IL was also investigated by observed wavelength of fluorescnece maxima. CcCel6A dissolved in a hydrated IL composed of dhp anions led to the development of a refolded state compared to that composed of bromide anions. To obtain the recovered protein in the buffer solution, aggregated CcCel6A was dissolved in hydrated IL, separated using a phenyl column, and collected in a buffer solution. The CcCel6A activity in each collected volume was measured using resorufin cellobioside. Aggregated CcCel6A dissolved in [N4444][dhp] separated on a phenyl column showed good activity levels than that with water soluble CcCel6A.