重组人肿瘤坏死因子受体Ⅰ蛋白复性过程中巯基浓度的监测  

Monitoring of free thiol concentration during refolding of recombinant human tumor necrosis factor receptorⅠprotein

作　　者：王保成[1] 李坤[1] 李春阳[1] 蔡峰[1] 秦娇荣 

机构地区：[1]成都生物制品研究所有限责任公司重组药物研究室,四川成都610023

出　　处：《中国生物制品学杂志》2013年第11期1646-1651,共6页Chinese Journal of Biologicals

基　　金："十二五"科技部重大新药专项基金:原创性人源化单克隆抗体药物研发技术平台(2011ZX09506-007)

摘　　要：目的建立一种在重组人肿瘤坏死因子受体Ⅰ（tumor necrosis factor receptorⅠ，TNFRⅠ）蛋白复性过程中简单、可靠且易于操作的巯基浓度的监测方法，用以指导、监控蛋白复性过程及终点判断。方法通过测定3种不同巯基浓度检测方法的线性范围及相关系数，建立适合重组人TNFRI蛋白复性溶液中巯基浓度的测定方法；动态监测蛋白复性过程中溶液巯基浓度变化及巯基浓度与复性蛋白生物活性的相关性；于蛋白复性48、96和144h取样，检测4℃保存3d和6d的稳定性。结果采用的3种巯基浓度监测方法中，方法3标准偏差值（standard error value，STDEV）较小，线性范围在0～12．5mmol／L，线性关系较好，R^2=0．9999，更适于蛋白复性溶液中巯基浓度的监测；随着蛋白复性时间的延长，巯基浓度呈明显的下降趋势（R^2=0．9891），当浓度在2～1mmol／L下降期间，是复性蛋白细胞活性上升最快期，而当巯基浓度降至1mmol／L及以下时，细胞活性达到最大值；蛋白复性144h后，细胞活性较为稳定。结论建立了一种简单、可靠且易于操作的巯基浓度测定方法，该方法对提高包涵体的复性效率具有重要的参考价值。Objective To develop a simple and reliable method for monitoring the free thiol concentration during refold-ing of recombinant human tumor necrosis factor receptor Ⅰ （TNFR Ⅰ ） protein to guide and monitor the refolding process and the judgment of endpoint. Methods The linear ranges and correlation coefficients of three methods were determined, based on which a method suitable for determination of free thiol concentration in refolded TNFR I solution was developed. The thiol concentration in TNFR I during refolding as well as its correlation to the biological activity of refolded protein were monitored by the method. Samples were taken from protein 48, 96 and 114 h after refolding and evaluated for stabil-ity after storage at 4 ~C for 3 and 6 d. Results The standard error value （STDEV） of test results by method 3 was the lowest in the three methods, while the linear range was 0 - 12. 5 mmol/L, R^2 = 0. 999 9, indicating that the method was more suitable for monitoring the thiol concentration in refolded protein solution. The thiol concentration showed a sig-nificantly decreasing tendency with the increasing hours for refolding （R^2 = 0. 989 1 ）. The biological activity of refolded protein increased rapidly during decrease of thiol concentration from 2 to 1 mmol / L, while reached a peak value when the thiol concentration decreased to 1 mmol / L or below. The cell activity was stable 144 h after refolding of protein. Conclusion A simple and reliable method for determination of thiol concentration was developed, which provided an im-portant reference for increasing the refolding efficiency of inclusion body.

关 键 词：蛋白质复性 巯基 浓度 细胞活性 

分 类 号：Q816[生物学—生物工程]