[Refolding of reduced/denatured RNase A the hydrophobic liquid-solid interface].
The renaturation of the reduced/denatured RNase A on the hydrophobic liquid-solid interface was investigated using hydrophobic interaction chromatography (HIC). The effects of urea concentrations, the ratios of reduced and oxidized glutathiones (GSH and GSSG), the pH of mobile phase and protein concentrations on the refolding efficiency and mass recovery of the reduced/denatured RNase A were investigated in detail. The results indicated that the reduced/ denatured RNase A can be refolded completely under the optimized conditions of pH 8.0, 2.0 mol/L urea and the concentration ratio of GSH/GSSG of 8: 1 in mobile phase. When the denatured protein was at the concentration of 5.0 mg/mL, the bioactivity efficiency and mass recoveries were 98.0% and 61.9% for 8.0 mol/L urea-denatured RNase A, respectively; and 100.1% and 66.8% for 7.0 mol/L guanidine hydrochloride (GuaHCl)-denatured RNase A, respectively. It proves that HIC is a powerful tool and new approach for protein refolding.
Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, Institute of Modern Separation Sciences, Key Laboratory of Separation Science in Shaanxi Province, Northwest University, Xi'an 710069, China.
Jing
J
Bi
Quan
Q
Bai
Jun
J
Wang
Lili
L
Wang
D002853:Chromatography, Liquid / Q000379:methods*; D057927:Hydrophobic and Hydrophilic Interactions; D011489:Protein Denaturation; D058849:Protein Refolding; D012259:Ribonuclease, Pancreatic / Q000737:chemistry*
D012259:Ribonuclease, Pancreatic