人工伴侣辅助溶菌酶复性的影响因素

Studying of the Effect Factors of Lysozyme Refolding Assisted by Artificial Chaperones

为了获得人工伴侣辅助蛋白质复性方法的广泛应用,本文以二硫苏糖醇还原、盐酸胍变性的溶菌酶为目标蛋白质,优化复性过程中的一些条件。首先确定了溶菌酶浓度为0.06mg/mL时,复性的最佳氧化还原体系GSSG/GSH均为3 mmol/L;然后证明了要达到较高的复性收率,在水浴环境下,需要较长的时间和较大的β-CD浓度(24 h,CTAB/β-CD为1.2mmol/L:14.4 mmol/L),收率可达90%以上。而在摇床环境下,利用较短时间和较低的β-CD浓度(1 h,CTAB/β-CD为1.2 mmol/L:4.8 mmol/L)就可以达到一定的复性收率(70%以上);进一步实验证明,若想在1 h内达到理想的复性效果,可以选用1.2 mmol/L:4.8 mmol/L的CTAB/β-CD,摇动10 min,然后保持静止,可达到70%以上的收率。

To make the way of getting lysozyme refolding assisted by artificial chaperones widely applied,we try to optimize the conditions of the refolding.The hydrochloric acid-Denatured and DTT-Reduced Lysozyme are taken as the model protein.Firstly,it is made clear that when the lysozyme concentration is 0.06 mg/mL,the GSSG and GSH to maximum refolding yield are both 3 mmol/L;then we prove that in the water-bath the higher refolding yield is achieved with longer time and higherβ-CD（24 h,CTAB/β-CD is 1.2 mmol/L：4.4 mmol/L）,the refolding yield can be over 90%.While,under the shaking conditions,a comparatively higher refolding yield（over 70%） could be obtained with shorter time and lowerβ-CD（1 h,CTAB/β-CD is 1.2 mmol/L：4.8 mmol/L）;In further experiment, we found the ideal refolding effect can be achieved within 1 h by shaking for 10 min and then keeping still, the refolding yield can be over 70%when CTAB/β-CD is 1.2 mmol/L：4.8 mmol/L.