应用分子伴侣共表达系统表达pfu 基因及酶活性测定

doi:10.3724 SP.J.1143.2009.09183

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

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 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

收稿日期:2009-09-14 出版日期:2009-12-25 发布日期:2009-12-25

Expression and Enzyme Activity Assay of pfu with Molecular Chaperone

ZHANG Hai-Jun¹ , YANG Jun^{2 , 3} , LIU Xiao-Guang¹ , HU Xiang-Yang^{2 , 3}

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

摘要/Abstract

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

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

中图分类号:

Q 78

张海军1 , 杨君2 , 3 , 刘晓光1 , 胡向阳2 , 3 . 应用分子伴侣共表达系统表达pfu 基因及酶活性测定[J]. Plant Diversity, 2009, 31(06): 499-503.

ZHANG Hai-Jun , YANG Jun^, , LIU Xiao-Guang , HU Xiang-Yang^,. Expression and Enzyme Activity Assay of pfu with Molecular Chaperone[J]. Plant Diversity, 2009, 31(06): 499-503.

[1]	杜维, 丁勇, 朱东阳, 尹继庭, 刘小烛, 赵平. 樟叶越桔熊果苷合成酶基因VdAS1的克隆及序列分析[J]. Plant Diversity, 2015, 37(01): 71-77.
[2]	于丽霞, 李斌, 黄海泉, 鄢波. 竹亚科lea3基因的进化分析[J]. Plant Diversity, 2014, 36(06): 707-714.
[3]	殷宪伦1、2, 王春涛1 , 孔祥翔1, 杨永平1, 胡向阳1. 利用TA克隆的方法简便构建入门克隆[J]. Plant Diversity, 2012, 34(4): 397-.
[4]	曾幼玲, 幸婷, 蔡忠贞, 张富春. 盐生植物盐爪爪甜菜碱醛脱氢酶基因的克隆及在盐胁迫下的BADH 基因的表达[J]. Plant Diversity, 2007, 29(01): 79-84.
[5]	杨涛，曾英. 植物萜类合酶研究进展[J]. Plant Diversity, 2005, 27(01): 1-10.
[6]	刘继梅　鄢波　郑丽屏　杜云龙　王玲仙　黄兴奇　 . 不同花色矮牵牛细胞色素b5蛋白的cDNA克隆及序列分析[J]. Plant Diversity, 2002, 24(02): 1-3.
[7]	郡波,马志刚,黄兴奇,王铃仙,胡忠. 美洲商陆一种抗真菌蛋自的cDNA克隆及序列分析[J]. Plant Diversity, 1998, 20(03): 1-3.
[8]	刘继梅；陈善娜；鄢波；黄兴奇；杨明挚. 水稻中编码甘油-3-磷酸转酰酶部分cDNA的克隆及序列分析[J]. Plant Diversity, 1998, 20(03): 1-3.

应用分子伴侣共表达系统表达pfu 基因及酶活性测定

Expression and Enzyme Activity Assay of pfu with Molecular Chaperone

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 8

编辑推荐

Metrics

本文评价