巯基微球协助重组γ-猪肝酯酶包涵体体外复性

Refolding of recombinant pig liver esterase inclusion bodies in vitro assisted by thiol-carrying latex particles

猪肝酯酶(PLE)是一种广泛应用于光学活性醇或羧酸合成的水解酶。大肠杆菌异体表达为大规模生产猪肝酯酶γ同工酶提供了可能,但表达过程中会形成包涵体,需经体外重折叠恢复其生物活性。针对γ-PLE含二硫键的特点,制备了三种不同巯基负载量的功能聚合物微球SG-DTT(6.1μmol·g-1、25.2μmol·g-1和143.4μmol·g-1),将其应用于协助γ-PLE包涵体体外复性,并考察了微球浓度、pH、温度和尿素浓度对复性效果的影响。复性过程需要适宜的氧化还原环境,较高的巯基负载量微球协助复性效果较好,当γ-PLE浓度为1000μg·ml-1时,微球协助复性后γ-PLE的活性可达到1885 U·L-1,比对照组提高了72%。研究表明,巯基微球能有效促进二硫键的正确配对,阻止蛋白分子间的聚集,是一种新型高效的复性添加剂。

Pig liver esterase （PLE） has been widely used in many fields, such as kinetic resolutions, desymmetrizations of prochiral substrates, and synthesis of nucleosides. Recombinant expression of the γ-isoenzyme of PLE （γ-PLE） in Escherichia coli can avoid the undesirable presence of several PLE isoenzymes in commercial production and interfering influence of other hydrolases, but y-PLE expressed in Escherichia coli is usually in the form of insoluble inclusion bodies which should be refolded in vitro to recover its biological activity. Three kinds of thiol-carrying latex particles SG-DTT with different concentrations of sulphydryl groups （6.1 μmol·g^-1, 25.2 μmol·g^-1 and 143.4 μmol·g^-1） were synthesized, and then used to assist refolding of γ-PLE in vitro. The effects of particle concentration, pH, refolding temperature and urea concentration on the refolding process were investigated. It was shown that refolding should be carried out under suitable redox environment and particles with higher concentration of sulphydryl groups were more effective. The final γ-PLE activity could achieve as high as 1885 U · L^-1 when γ-PLE concentration was 1000 μg·ml^-1, increased by 72% than that without the particles. All results above demonstrated that thiol-carrying latex particles could assist y-PLE refolding by attacking disulfide bonds in misfolded protein to form correct pairing through thiol/disulfide exchange reactions and inhibiting intermolecular interactions, which presented an alternative way to facilitate refolding of proteins with disulfide bonds in vitro.