摘要
目的探讨大肠杆菌表达的卵巢癌抗独特型单链抗体/小鼠热休克蛋白70(6B11ScFv/mHSP70)融合蛋白包涵体变性、复性条件,以确定其最佳体外复性条件。方法大肠杆菌表达大量融合蛋白6B11ScFv/mHSP70:①比较不同的裂解液(8mol/L尿素和6mol/L盐酸胍)对包涵体的裂解效率及其对复性蛋白活性的影响;②探索序贯稀释方法,以及氧化还原环境和复性液中不同浓度L-精氨酸对蛋白稀释复性的影响;③比较稀释复性和柱上复性对融合蛋白复性效率及活性的影响。采用Bradford法测定包涵体裂解液与复性后蛋白浓度。所有复性蛋白活性的检测均采用ELISA方法。结果以包涵体形式表达6B11ScFv/mHSP70蛋白:①经6mol/L盐酸胍裂解包涵体的效率远高于8mol/L尿素,但前者复性所得蛋白活性低,6mol/L盐酸胍彻底裂解的包涵体经8mol/L尿素透析后再复性,复性效率显著提高;②序贯稀释方法可提高蛋白复性效率;加入0.5mol/LL-精氨酸可降低稀释复性过程中蛋白聚集物的产生,提高复性效率;加入还原型谷胱甘肽(GSH)与氧化型谷胱甘肽(GSSH)浓度比为1:5时,可提高复性后蛋白活性;③柱上复性可一步完成蛋白的复性和纯化,所得蛋白纯度较高,但复性效率和复性后蛋白活性较稀释复性低。结论本研究建立了6B11ScFv/mHSP70融合蛋白的最佳复性条件:包涵体经6mol/L盐酸胍彻底裂解后,再经8mol/L尿素透析,然后进行序贯稀释复性,且复性液中加入0.5mol/LL-精氨酸和GSH:GSSH=1:5以提高复性效率和蛋白活性,为进一步研究该蛋白的功能奠定了基础。
Objective To explore and to determined the optima conditions for refolding of the prokaryotic 6B 11ScFv/mHSP70 fusion protein in vitro.Methods To express the recombinant 6B11ScFv/mHSP70 fusion protein by the prokaryotic expression system E.coli： （1） To compare the efficiency of 8 mol/L urea and 6 mol/L guanidine hydrochloride in solution of the inclusion body; （2） To examine the effects of sequential dilution method, presence of different redox system and diverse L-arginine concentration on protein aggregation and activity during the refolding process; （3） To investigate the value of dilution refolding and on-column refolding in the recovery of active 6B 11ScFv/mHSP70 protein. The concentrations of protein in both inclusion body lysate and refolding buffers were determined by Bradford method. We evaluate the activity of this fusion protein to combine with specific antibody by ELISA.Results The recombinant proteins were expressed in the form of inclusion body in E.coli BL21（DE3）： （1） The solution efficiency of 6 mol/L guanidine hydrochloride was much higher than that of 8 mol/L urea. But the former protein activity after refolding was poorer than the latter. To dissolve the inclusion body in 6 mol/L guanidine hydrochloride and then dialyze with 8 mol/L urea before the refolding process exhibits excellent efficiency and protein activity; （2） The sequential dilution procedure and the addition of 0.5 mol/L L-arginine could effectively reduce protein aggregation in the dilution process. The addition （GSH：GSSH = 1：5） may facilitate refolding but not suppress aggregation; （3） The on-column refolding could achieve both recovery and purification of the protein in a single procedure but with lower refolding efficiency and poorer activity than dilution refolding for this protein. Conclusions We establish the optimal solution and refolding conditions for the 6B11ScFv/mHSP70 fusion protein. The inclusion body of the fusion protein was dissolved thoroughly in 6 mol/L guanidine hydrochloride, and then dialyzed with 8 mol/L urea. The dissolved extracts were refolding in buffers with 0.5 mol/L L-arginine and GSH：GSSH = 1：5 by sequential dilution. And the refolding protein shows satisfactory immunoactivity and could be used in further research of protein functions.
出处
《中国医药生物技术》
CSCD
2009年第6期424-429,共6页
Chinese Medicinal Biotechnology
基金
国家自然科学基金(30772320)
