Ribonuclease A Inclusion Body Refolding Affected By Redox System

In renaturation process of disulfide-containing protein, the formation of nature disulfide bonds is usually the rate-limiting step of renaturation process; wrong formation of disulfide bonds causes protein aggregation, which is the key reason of decrease of renaturation yield.To develop a method that assists disulfide-containing protein with efficient renaturation, a novel redox system (ArSH/HCA) composed of two kinds of small-molecule is set up in this study, and they are 4-mercaptophenylacetic acid (ArSH) and hexanoyl cystamine (HCA) respectively, both of which are able to catlalyze two disulfide bonds to generate quickly. This system is omptimized with RNase A as a protein model to determine the optimal condation for RNase A renaturation; finally, RNase A inclusion body is renatured under the optimum condation determined, and the results are compared with that from traditional redox system.The best renaturation condation determined through optimizing is in pH8.3 Tris- acetate solution that contains 0.4M urea,1 mM EDTA,1 mM ArSH and 0.25 mM HCA, which makes 0.3 mg·mL-1RNase A get renaturation balance in 3 h whose renaturation yield finally achieves 92%. While the same concentration of RNase A achieves renaturation balance in 6 h with renaturation yield 65%, asistd by traditional redox system (1 mM GSH/0.25 mM GSSG), other condation being equal.Based on the former study and combied with the actual situation, the renaturation condation undergoes further optimizaion and reaches the optimum, namely, in pH8.3 Tris- acetate solution that contains 1 M urea,1 mM EDTA,4 mM ArSH and 0.5 mM HCA, which makes 0.3 mg·mL1 mM GSH/0.25 mM GSSG), other condation being equal. -1RNase A inclusion body get renaturation yield as high as 50% in 1 h, and finally reaches balance after 5 h with high renaturation yield 70%. RNase A inclusion body hardly renaturates with renaturation yield as low as 35% under the same condation assisted by GSH/GSSG or GSH/HCA; though it can get renaturation yield as high as 60% assisted by ArSH/GSSG, the renaturation balance needs 7 h. The approach developed in this study gets significant improvement compared with former work; RNase A concentration is enhanced from 0.12 mg mL-1 to 0.3 mg mL-1; time for renaturation balance is compressed from 24 h to 5 h; renaturation yiesd is enhanced from 50% to 70%; all these results show inclusion body is basically able to renaturate with high effiecncy, high concentration, high velocity.