文献类型：期刊文献

中文题名：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

英文题名：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：[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

年份：2012

卷号：70

期号：4

起止页码：453

中文期刊名：化学学报

外文期刊名：Acta Chimica Sinica

收录：CSTPCD;;Scopus;WOS:【SCI-EXPANDED(收录号:WOS:000301637400014)】；北大核心:【北大核心2011】；CSCD:【CSCD2011_2012】；

基金：National Key Basic Research Program of China (No. 2009CB918600).

语种：中文

中文关键词：核磁共振;稳态荧光;ANS荧光探针;MMP-12;复性折叠

外文关键词：NMR; steady-state fluorescence; ANS; MMP-12; refolding

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.