用疏水色谱对还原型胍变性牛胰岛素的折叠特性研究

用疏水相互色谱(HPHIC)对还原胍变性牛胰岛素在疏水界面上的折叠与复性进行了研究.结果表明,采用普通流动相时,对还原胍变胰岛素的复性效果较差,而采用氧化型流动相可使其复性效率提高到66%,并用反相色谱(RPLC)、紫外吸收光谱、荧光光谱及MALDI-TOF对其复性效果进行了验证.同时与体积排阻色谱(SEC)和稀释法对还原胍变胰岛素的复性结果进行了比较.结果表明,SEC根本无法使还原胍变胰岛素复性,而稀释法的复性效率仅有2%.这进一步表明HPHIC是变性蛋白复性的有效工具,变性蛋白在疏水界面折叠过程中,蛋白质与固定相之间的疏水相互作用对蛋白折叠起着关键性的作用,是蛋白折叠的主要驱动力.

Studies on the Refolding of Reduced-Denaturated Insulin with High Performance Hydrophobic Interaction Chromatography

The refolding or renaturation of reduced denaturated Insulin from bovine pancreas with 7.0 mol/L guanidine hydrocloride(GuaHCl) solution on a moderate hydrophobic surface was studied with a high performance hydrophobic interaction chromatography(HPHIC). It showed that the reduced denaturated Insulin could be refolded partly with HPHIC when the general mobile phase was used in HPHIC . However, in the presence of oxidized glutathione(GSSG) in the mobile phase employed, the disulfide exchange of reduced denaturated Insulin can be accelerated, and the most of three disulfide bridges of Insulin were formed correctly. The result indicates that the refolding yield of reduced denaturated Insulin with GuaHCl should be increased up to 66%. The result was further tested by the separation of the HPHIC fractions of reduced denaturated Insulin with reversed phase high performance liquid chromatography(RPLC), the UV spectra, the flourescence spectra and MALDI TOF. In addition, compared to the refolding yields of reduced denaturated Insulin renaturated with size exclusive chromatography(SEC) and dilution, it showed that although GSSG was used in the mobile phase, the reduced denaturated Insulin can not be refolded with SEC at all. The refolding yield of it with dilution is only less than 2%. It proved further that during the refolding process of unfolded protein refolded on the hydrophobic surface, the interactions between Insulin and the HPHIC stationary phase is very critical and may be the drive force for protein refolding and HPHIC may be the potential refolding tool of unfolded proteins.