Studies On Efficient Methods For Ribonuclease A Refolding From Inclusion Bodies

Because of the slow formation of disulfide bonds and the extensive proteinaggregation, refolding ribonuclease A (RNase A) from IBs at high proteinconcentration was of low efficiency and limited its large-scale preparation. Herein,efficient refolding methods were studied to facilitate the refolding of RNase A fromIBs, by improving the formation of disulfide bonds and suppressing the proteinaggregation.In order to improve the formation rate of disulfide bonds in RNase A, a newredox system composed of4-mercaptobenzeneacetate (ArSH) and hexanoylcystamine (HCA) was proposed. It was found that the oxidative refolding of0.3mg/mL RNase A from commercial source reached a refolding yield of94%in2hwith ArSH/HCA at optimized concentrations, which was two times faster than thatwith traditional redox agents such as reduced glutathione (GSH) and oxidizedglutathione (GSSG). Moreover, the refolding yield of0.3mg/mL RNase A from IBs(0.46mg/mL in total protein concentration) could also reach89%in3h withArSH/HCA, much better than the performance with GSH/GSSG, leding to over60%reduction in the refolding time in the inclusion body protein refolding. These resultsindicated that the new redox system was definitely effective for the practical oxidativerefolding of RNase A from IBs.When the refolding of RNase A from IBs was carried out at elevatedconcentrations, the refolding yield was compromised by the protein aggregation. Ithas been found that like-charged medium could facilitate protein refolding byinducing the oriented alignment of protein molecules near the charged surface andleading to the inhibition of protein aggregation. Herein, the like-charged mediumswith different charge densities were prepared to assist the refolding of RNase A fromIBs at elevated concentrations. As a result,0.9mg/mL RNase A (1.4mg/mL in totalprotein concentration) could be63%refolded, which was a29%increase as comparedto that without the additive. What’s more, at low additive concentration range, themedium of high charge density could resulted in a distinct increase in refolding yield,which is beneficial in facilitating the protein refolding.Moreover, coupling with the new redox system, the artificial chaperone (AC)containing cetyltrimethylammonium bromide (CTAB) and β-cyclodextrin (β-CD) has been used to refold RNase A from IBs at elevated concentrations. At low ureaconcentration (0.8mol/L), the AC could enhance the refolding yield of RNase A byeffectively suppressing its intermolecular interaction-induced aggregation. As a result,0.9mg/mL RNase A (1.4mg/mL in total protein concentration) could be77%refolded, which was a57%increase as compared to that without the AC. At highprotein concentration range and1.6mol/L urea, CTAB selectively precipitatedcontaminant proteins distinctly, so a purification effect was achieved. For example,1.5mg/mL RNase A (2.3mg/mL in total protein concentration) could be62%refolded at a purity of87%, which was a34%increase in purity as compared to that inIBs (65%). In summery, AC with anti-aggregation and purification effect led to theefficient preparation of RNase A from IBs at elevated protein concentration.