中山大学学报自然科学版 ›› 2011, Vol. 50 ›› Issue (5): 93-99.

• 研究论文 • 上一篇    下一篇

交叉酶切联合MALDI-TOF/MS分析重组蛋白二硫键分布的方法建立

徐祖敏1,程 锐1,杨 霞1,杨中汉1,刘少军2,黎明涛2,蔡卫斌1,高国全1   

  1. (1. 中山大学中山医学院生化教研室,广东 广州 510080;2.中山医学院蛋白质组学实验室,广东 广州 510080)
  • 收稿日期:2010-12-07 修回日期:1900-01-01 出版日期:2011-09-25 发布日期:2011-09-25
  • 通讯作者: 蔡卫斌

Unraveling Disulfidebond Distribution of Recombinant Peptide by Orthogonal Protease Digestion and MALDITOF/MS

XU Zumin1, CHENG Rui1, YANG Xia1, YANG Zhonghan1, LIU Shaojun2, LI Mingtao2, CAI Weibin1, GAO Guoquan1   

  1. (1.Department of Biochemistry and Molecular Biology,Sun Yatsen University, Guangzhou 510080, China;〖JP〗 2. Proteomics Laboratory, Zhongshan Medical School, Sun Yatsen University, Guangzhou 510080, China)
  • Received:2010-12-07 Revised:1900-01-01 Online:2011-09-25 Published:2011-09-25

摘要: 以Kringle环为分子模型,应用MALDI-TOF/MS联合特异性蛋白酶酶切技术,探讨建立蛋白质二硫键分布和定位分析的研究方法。采用pET22b(+)原核表达体系诱导表达重组人纤溶酶原K5蛋白(rhK5),经Ni2+-NTA resin亲和层析纯化,并通过SDSPAGE和Westernblot进行初步鉴定;采用MTT法分析rhK5对微血管内皮细胞的抑制活性;联合应用交叉酶切(Trypsin Gold单切或Trypsin Gold及Endoproteinase ASPN双酶切)和基质辅助激光解析-飞行时间质谱(MALDITOF/MS)分析rhK5的二硫键分布;并应用生物信息学方法和圆二色谱法进一步验证rhK5二级结构。应用原核体系高效表达具有生物活性的rhK5蛋白,经交叉酶切联合MALDI-TOF/MS证实rhK5中的6个半胱氨酸正确配对形成了3对二硫键(Cys462:541, Cys483:524和Cys512:536),圆二色谱法测定发现rhK5为典型的β折叠型结构。上述研究结果为分析基因工程重组蛋白质中半胱氨酸配对及二硫键分布提供了方法学基础。

关键词: 基因工程, 重组蛋白, 蛋白质结构, 二硫键, 基质辅助激光解析电离飞行时间质谱

Abstract: In order to establish a method for analyzing the distribution and localization of disulfide in recombinant protein by specific protease digestion combined with MALDITOF/MS. The pET22b (+) prokaryotic system was harnessed to generate recombinant protein of human plasminogen kingle (rhK5) which was purified by Ni2+-NTA resin affinity chromatography. SDS-PAGE and Western blot analysis were used to identify the recombinant protein. The disulfide bridging conformation and dimensional structure of rhK5 were predicted by 3DJIGSAW Comparative Modeling Server (UK) and the disulfide bond distribution of rhK5 was confirmed by orthogonal enzymatic digestion and MALDITOF/MS. The secondary structure of rhK5 was analyzed by circular dichroism. Enzymatic digestion and MALDI-TOF/MS analysis demonstrated the presence of disulfide bridges among Cys462Cys541, Cys483Cys524 and Cys512Cys536 in rhK5. Circular dichroism showed that rhK5 adopted the shape characteristic of a β-sheet. Our studies provide methodological basis for analyzing the pairing and distribution of disulfide bond of genetic engineering recombinant proteins.

Key words: genetic engineering, recombinant protein, protein structure, disulfide bond, matrixassisted laser desorptiontime of flight/ionization mass spectrometry (MALDI-TOF/MS)

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