脲变性α-糜蛋白酶的高效液相色谱复性

Refolding of urea-denatured α-Chy by high performance hydrophobic interaction chromatography

目的研究变性蛋白质在高效疏水作用色谱(HPHIC)固定相表面上的保留行为及活性回收率。方法用脲将标准蛋白质α-糜蛋白酶(α-Chy)变性,然后以硫酸铵为流动相,用HPHIC固定相(TSK)对脲变性α-Chy进行复性,并测定复性α-Chy的Z值、质量及活性回收率。结果Z值随脲浓度的增大而减小,变性剂浓度在1.0～3.0mol·L-1范围内时,HPHIC对变性α-Chy有较高的活性回收率。结论HPHIC是变性蛋白复性的有效工具,蛋白质与固定相之间的疏水作用是蛋白折叠的主要驱动力。

Objective To investigate the efficiency of renaturation and retention behavior of denatured proteins by high performance hydrophobic interaction chromatography（HPHIC）. Methods The standard protein, α-Chymotrypsin （α-Chy）, was denatured by urea, and then it was renatured by HPHIC in mobile phase of ammonium sulfate. Z value, mass and bioactivity recovery were detected. Results The Z value was decreased dramatically with an increase in the concentration of urea. The bloatrive recovery of α-Chy was high when α-Chy was denatured by urea with the concentration ranging from 1.0 to 3.0 mol·L^-1. Conclusion High performance hydrophobic interaction chromatography plays an efficient tool in the protein renaturation and the interaction between protein and the HPHIC stationary phase is the driving force for protein refolding.