温敏型聚合物PNIPAAm辅助的溶菌酶体外复性

TEMPERATURE SENSITIVE PNIPAAm ASSISTED PROTEIN REFOLDING in vitro

合成了 3种具有不同分子量的温敏型聚合物聚 (N 异丙基丙烯酰胺 ) (PNIPAAm) ,测定了其分子量分布以及相应的低临界溶解温度 (LCST) .在溶菌酶复性溶液中加入PNIPAAm可促进溶菌酶复性 ,其中采用中等分子量M—PNIPAAm(Mw 为 2 2× 10 4 g mol)时溶菌酶的复性效果最佳 ,并采用荧光发射光谱技术表征了PMIPAAm分子结构对于溶菌酶结构的影响 .系统考察了采用M—PNIPAAm时 ,复性液中尿素浓度、蛋白质浓度和温度等条件对溶菌酶复性效果影响 .结果显示尿素与M—PNIPAAm对于溶菌酶复性呈现协同效应 ,复性操作温度不仅同溶菌酶自身特性有关 ,而且还受到M—PNIPAAm自身性质变化的影响 .

Temperature sensitive polymers, poly-N-isopropyl acrylamides (PNIPAAm) with different molecular weights, were prepared by radical polymerization. The molecular weight distribution and lower critical solution temperature of these polymers were deter mined by GPC and spectrophotometry. The feasibility of the polymer in assisting the refolding of denatured lysozyme was examined, by directly diluting the denatured lysozyme into refolding buffer containing PNIPAAm of different molecular weight. PNIPAAm with the mean molecular weight of 22, 000 (M_ PNIPAAm) showed the best function of assisting lysozyme refolding, as indicated by the recovery of lysozyme activity from the denatured lysozyme sample. The molecular interaction of PNIPAAm with lysozyme and its implication on the structure change of lysozyme were investigated by fluorescence emission spectroscopy. The effects of lysozyme concentration and temperature on the lysozyme refolding were examined and the synergy of urea and MPNIPAAm on lysozyme refolding was identified. The suitable temperature for lysozyme refolding was chosen according to the temperature responsive behavior of PNIPAAm. The results shown by present study showed the high potential of temperature sensitive polymers in high concentration protein refolding in vitro in large scale.