温敏型聚合物PNIPA和P(NIPA-co-SA)的制备及其协助溶菌酶复性研究

Synthesis of Thermo-Sensitive Polymers of PNIPA and P(NIPA-co-SA) and their Application in the Refolding of Denatured Lysozyme in vitro

采用反相悬浮法合成了温敏型聚合物聚N-异丙基丙烯酰胺(PNIPA)凝胶及其与丙烯酸钠(SA)的共聚凝胶P(NIPA-co-SA),对凝胶的形态、粒径分布、表面特性以及温敏性能进行了考察,共单体丙烯酸钠的引入使得凝胶的最大溶胀倍率和低临界溶解温度(LCST)都有明显的提高。将两种凝胶用于目标蛋白溶菌酶的体外复性过程,考察了其协助复性的效果。结果表明:当蛋白浓度为250μg·mL-1时,加入80mg·mL-1PNIPA凝胶可使溶菌酶的活性回收率由稀释复性的51.3%提高到72.9%;加入120mg·mL-1P(NIPA-co-SA)共聚凝胶可使活性回收率达到71.5%。溶菌酶的浓度越高,与稀释复性相比凝胶协助复性的效果就越好。复性后凝胶可方便地分离回收,PNIPA凝胶重复使用6次后,溶菌酶的活性回收率仍高于稀释复性15%以上。

The thermo-sensitive polymers of PNIPA and P（NIPA-CO-SA） gel particles were synthesized by inverse suspension polymerization. The morphology, size distribution and thermo-sensitive characteristics of the prepared gel particles were studied, and it was found that the maximal swelling ratio and lower critical solution temperature （LCST） of the prepared gel particles increase obviously with the addition of sodium acrylate （SA） monomer. The effects of the gels for assisting the refolding of denatured lysozyme were then examined; when the protein concentration is 250 ~tg-mL-~, the found optimum concentration of PNIPA and P（NIPA-CO-SA） gel used for assisting the refolding process are 80 lag.mL-1 and 120 lag.mL-l, respectively, and using the above optimum concentrations, the activity recovery of lysozyme increases from 51.3% （when using simple dilution refolding） to 72.9% （refolding assisted by PNIPA gel particles） and 71.5% （refolding assisted by P（NIPA-CO-SA gel particles）, respectively. The further results indicate that the higher initial denatured lysozyme concentration, the assisting effect of the prepared gel particles for refolding is more efficient. After refolding, the gel particles could be removed easily; and after reused for 6 times, the activity recovery of lysozyme refolding by reused PNIPA gel particles can still be 15% higher than that refolding by simple dilute refolding.