摘要
通过测定复性后酶活性和计算蛋白复性收率,研究了L-精氨酸助溶菌酶的最佳复性条件,利用蛋白质的复性和聚集反应竞争三态动力学模型描述了L-精氨酸助溶菌酶复性动力学,在溶菌酶浓度为100~500μg.mL-1、L-精氨酸浓度为0~1.0 mol.L-1的条件下,分析了溶菌酶浓度及L-精氨酸浓度对复性动力学常数的影响。结果表明,L-精氨酸助溶菌酶复性符合三级聚集反应动力学,L-精氨酸的主要作用是抑制蛋白聚集体的生成速率,从而达到抑制蛋白沉淀、提高复性收率的效果。
Protein refolding is an important operation in the down-stream processing of many genetically engineered proteins.L-Arginine is one of the most commonly used additives that are effective in assisting refolding of recombinant proteins from inclusion bodies.A major problem of protein refolding is aggregation of an unfolded or an intermediate state during refolding.L-Arginine may work on refolding of proteins by suppressing aggregation of proteins,thereby allowing the unfolded or intermediate state to proceed to the native state.The mechanism behind its action is still not fully understood.Using lysozyme as a model protein,the optimal refolding conditions of lysozyme refolding facilitated by L-arginine were investigated.A kinetic model based on the competition between protein folding and aggregation was employed to express the refolding process of denatured/reduced lysozyme facilitated by L-arginine.Within the range of 100～500 μg·mL-1 lysozyme,the effect of L-arginine concentration from 0 to 1.0 mol·L-1 on the refolding kinetics constants was investigated.It was found that the aggregation reaction of the L-arginine-assisted refolding could be described as a third order reaction.The primary role of the L-arginine was to suppress the aggregation reaction rate,resulting in the increasing of refolding yield.The observations suggest that L-arginine may suppress aggregation of the protein during refolding.
出处
《化学与生物工程》
CAS
2010年第10期50-54,共5页
Chemistry & Bioengineering
基金
上海市优秀青年教师科研基金资助项目(B37-0111-09-003)
