化学学报 ›› 2012, Vol. 70 ›› Issue (04): 453-458.DOI: 10.6023/A1109161 上一篇    下一篇

研究论文

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

张琳琳a,b,c, 冯延叶a,b, 蓝文贤c, 曹春阳c, 徐殿胜a, 杨忠a,b   

  1. a 华东理工大学生物反应器工程国家重点实验室 上海 200237;
    b 复旦大学生命科学学院微生物学和微生物工程系 上海 200433;
    c 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
  • 收稿日期:2011-09-16 修回日期:2011-10-18 出版日期:2012-02-28 发布日期:2012-03-07
  • 通讯作者: 杨忠
  • 基金资助:

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

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

Zhang Linlina,b,c, Feng Yanyea,b, Lan Wenxianc, Cao Chunyangc, Xu Dianshenga, Yang Zhonga,b   

  1. a State Key Laboratory of Bioreactor Engineering, School of Biotechnology, East China University of Science and Technology, Shanghai 200237, China;
    b Department of Microbiology and Microbial Engineering, School of Life Sciences, Fudan University, Shanghai 200433, China;
    c State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China
  • Received:2011-09-16 Revised:2011-10-18 Online:2012-02-28 Published:2012-03-07
  • Supported by:

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

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-15N HSQC (heteronuclear single quantum correlation)谱图显示, 尿素浓度从4 mol/L 降低到3 mol/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4 mol/L 到3 mol/L 的浓度变化缓慢), 实现复性收率提高一倍.

关键词: 核磁共振, 稳态荧光, ANS 荧光探针, MMP-12, 复性折叠

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 improve the refolding yield from inclusion bodies. The midpoint for the refolding (Cm≈4, mid-point of transition) of MMP-12 obtained from NMR experiments coincides with those from intrinsic tryptophan fluorescence 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.

Key words: NMR, steady-state fluorescence, ANS, MMP-12, refolding