Protein Liquid Chromatography Refolding

Protein renaturation is the most important technique in the downstream technology. In recent years, liquid chromatography has become a prosperous method for protein renaturation. The renaturation of proteins denatured on the different liquid chromatography was studied in this paper.Eleven proteins were denatured by three denaturing agents i.e. GuHCl, Urea and SDS. The proteins denatured were refolded by high-performance hydrophobic interaction chromatography(HPHIC) and high-performance ion-exchange chromatography (HPIEC). There are five proteins can be refolded on the HPHIC and three proteins on the HPIEC. The refolding efficiency on the HPHIC, such as mass recovery, and activity recovery were investigated. The results showed that the refolding efficiency of proteins denatured by GuHCl is the highest, and SDS is the lowest in the three denaturing agents. The similar results were showed on the HPIEC. But the mass recovery and activity recovery of proteins refolded on the HPIEC are lower than that of on the HPHIC. It is more difficult to select the refolding conditions on the HPIEC than on the HPHIC.In the second part, RNase of GuHCl denatured and urea denatured weredeoxidized by dithiotheitol(DTT), then were oxidized by oxidizedgluthatione (GSSG) and were refolded on HPHIC.HPIEC, and size exclusion chromatography (SEC). The mass recovery and activity recovery of RNase on the HPHIC and HPEC are higher than that on the SEC. The stationary phase of three chromatography all contribute to the refolding of RNase. The refolding efficiency of RNase can be increased by adjusting the ratio of oxidized agent to deoxidized agent or adding low concentration of the denaturing agents to refolding solution.