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Acta Chimica Sinica ›› 2012, Vol. 70 ›› Issue (04): 453-458.DOI: 10.6023/A1109161 Previous Articles     Next Articles

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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
  • 投稿日期:2011-09-16 修回日期:2011-10-18 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 Published:2012-03-07
  • Supported by:

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

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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