Integrative Biology Journals
Plant Diversity

Plant Diversity ›› 2009, Vol. 31 ›› Issue (06): 499-503.DOI: 10.3724 SP.J.1143.2009.09183

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Expression and Enzyme Activity Assay of pfu with Molecular Chaperone

ZHANG Hai-Jun1 , YANG Jun2 , 3 , LIU Xiao-Guang1 , HU Xiang-Yang2 , 3   

  1. 1 Institute of Life Sciences, Jiangsu University , Zhenjiang 212013 , China; 2 Kunming Institute of Botany,Chinese Academy of Sciences, Kunming 650204 , China; 3 Institute of Tibetan Plateau Research at Kunming, Chinese Academy of Sciences, Kunming 650204 , China
  • Received:2009-09-14 Online:2009-12-25 Published:2009-12-25

应用分子伴侣共表达系统表达pfu 基因及酶活性测定

张海军1 , 杨 君2 , 3 , 刘晓光1 , 胡向阳2 , 3   

  1. 1 江苏大学生命科学研究院, 镇江江苏 212013; 2 中国科学院昆明植物研究所, 云南昆明 650204;
    3 中国科学院青藏高原研究所昆明部, 云南昆明 650204

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Abstract: Co-expressing the plasmid pET32a- pfu which was constructed using Infution technique with chaperone plasmid HG-PGRO7 at the same time in E. coli . BL21 (DE3) . The expression system, which expressed pfu gene alone in E. coli . BL21 (DE3) , was used as control .Most of unnecessary proteins were exenterated by heat treatment and (NH4 )2 S04 precipitation. The purified fusion protein was obtained by the Ni chelating resin affinity chromatography. The SDS-PAGE analysis showed that the molecular mass of the purified fusion protein was about 90 kD, conforming to the expected molecular mass of Pfu protein. The results of enzyme activity assay of pfu demonstrated that molecular chaperone was able to activate pfu gene expression and its enzyme activity .

Key words: Pfu

摘要： 将通过In-fution 方法构建的pET32a- pfu 质粒与可以促进可溶性表达的HG-PGRO7 质粒一起转入大肠杆菌BL21 (DE3) 共表达, 以pET32a- pfu 单独在BL21 (DE3) 中表达作为对照。用热变性和(NH4 )2 SO4 沉淀去除部分杂蛋白, 再经Ni-NAT 亲和层析柱纯化分离pfu 蛋白, SDS-PAGE 检测结果表明目的蛋白大小约为90 kD, 与预计的分子量大小一致。最后对其酶活性测定结果表明分子伴侣能够促进pfu 基因表达, 并能够提高酶活性。

关键词: Pfu, 分子伴侣, 基因克隆, 载体构建, 原核表达, 蛋白纯化, 酶活测定

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