MMP-12蛋白包涵体复性时折叠状态核磁共振图谱与荧光光谱研究  

Investigation on MMP-12 Aggregation State in Urea Contained Solution by NMR and Fluorescence Spectroscopy

作　　者：张琳琳[1,2,3] 冯延叶[1,2] 蓝文贤[3] 曹春阳[3] 徐殿胜[1] 杨忠[1,2] 

机构地区：[1]华东理工大学生物反应器工程国家重点实验室,上海200237 [2]复旦大学生命科学学院微生物学和微生物工程系,上海200433 [3]中国科学院上海有机化学研究所生命有机国家重点实验室,上海200032

出　　处：《化学学报》2012年第4期453-458,共6页Acta Chimica Sinica

基　　金：国家重点基础研究发展计划(973计划)(No.2009CB918600)资助项目~~

摘　　要：MMP-12是癌症治疗药物靶标.为了更好研制新药,需要大量制备MMP-12,但MMP-12在大肠杆菌中以包涵体形式表达.因此如何优化蛋白复性过程是大量获取MMP-12蛋白的关键.采用核磁共振、稳态荧光法、外源性ANS(8-anilinol-naphthalenesulfonic acid)荧光探针三种方法监控MMP-12变性蛋白的再折叠过程,以探究其复性折叠机制.研究发现MMP-12再折叠中点值与对应的尿素浓度几乎相等(Cm≈4,mid-point of transition).不同尿素浓度中MMP-12的二维1H-15NHSQC(heteronuclear single quantum correlation)谱图显示,尿素浓度从4mol/L降低到3mol/L是MMP-12蛋白复性折叠的关键步骤.据此我们将MMP-12蛋白复性从常规的梯度透析复性方法改进成等容透析复性法(即确保尿素从4mol/L到3mol/L的浓度变化缓慢),实现复性收率提高一倍.MMP-12 is a drug target for cancer therapy,but it＇s over-expressed as inclusion bodies in E.coli To produce it in a large scale,in this paper,NMR,intrinsic tryptophan fluorescence,and ANS（8-anilinol-naphthalenesul fonic acid） fluorescence were employed to monitor its aggregation state to im-prove the refolding yield from inclusion bodies.The midpoint for the refolding（Cm≈4,mid-point of transi-tion） of MMP-12 obtained from NMR experiments coincides with those from intrinsic tryptophan fluores-cence and ANS fluorescence.These data suggest that the urea concentration from 4 mol/L to 3 mol/L is a key step in refolding process of MMP-12.Based on these results,we changed the refolding method from conventional step-wise dialysis to continuous dialysis,thus the refolding yield of MMP-12 from its inclusion bodies was doubled.

关 键 词：核磁共振 稳态荧光 ANS荧光探针 MMP-12 复性折叠 

分 类 号：Q51[生物学—生物化学]