人S100A2-GST融合蛋白原核表达和纯化

卫佳; 王胜; 苗静琨; 陈英华; 李星星; 吴丽美; 左国伟; 何通川; 周兰

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人S100A2-GST融合蛋白原核表达和纯化

论著 | 更新时间：2024-01-17

- 人S100A2-GST融合蛋白原核表达和纯化
- Expression and purification of human S100A2-GST fusion protein in prokaryotic cells
- 重庆医科大学学报 2008年33卷第12期页码：1427-1430
- 作者机构：
  
  1. 重庆医科大学生物医学工程系
  2. 美国芝加哥大学医学中心分子肿瘤研究室
- 作者简介：
- 基金信息：
  
  国家自然科学基金（30340039）;教育部“春晖计划”科研启动经费资助项目（2003年）;2004年重庆市留学回国人员启动基金
- DOI：
  中图分类号： Q786
- 纸质出版日期：2008，
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[1]卫佳,王胜,苗静琨,陈英华,李星星,吴丽美,左国伟,何通川,周兰.人S100A2-GST融合蛋白原核表达和纯化[J].重庆医科大学学报,2008,33(12):1427-1430.

WEI Jia, et al. Expression and purification of human S100A2-GST fusion protein in prokaryotic cells[J]. Journal of Chongqing Medical University, 2008,33(12):1427-1430.
[1]卫佳,王胜,苗静琨,陈英华,李星星,吴丽美,左国伟,何通川,周兰.人S100A2-GST融合蛋白原核表达和纯化[J].重庆医科大学学报,2008,33(12):1427-1430. DOI：

WEI Jia, et al. Expression and purification of human S100A2-GST fusion protein in prokaryotic cells[J]. Journal of Chongqing Medical University, 2008,33(12):1427-1430. DOI：

摘要

目的:制备人S100A2-GST融合蛋白（Human S100A2-GST fusion protein

hS100A2-GST）

为进一步研究人hS100A2蛋白的功能及其与其它因子或系统的相互作用奠定基础。方法:从pHAHA-hS100A2中双酶切获取hS100A2基因

亚克隆至原核表达载体pGST-moluc

构建pGST-moluc-hS100A2

转化BL21后酶切鉴定

IPTG诱导重组菌表达融合蛋白hS100A2-GST

再经过Glutathion-Sepharose4B球珠分离纯化、SDS-PAGE及Western Blot鉴定

Bradford法蛋白定量。结果:XhoⅠ和EcoRⅠ双酶切该重组质粒后获得2个片段

分别约300bp和5kb

提示pGST-moluc-hS100A2构建正确;重组菌株经过IPTG诱导后

表达分子量约为36kD的蛋白

产率达5mg/L菌液;Glutathion-Sepharose4B球珠纯化后

该融和蛋白纯度达92%;Western Blot显示该蛋白可以被S100A2的抗体特异识别

证实其为hS100A2-GST融合蛋白。结论:成功构建了hS100A2-GST原核表达质粒pGST-moluc-hS100A2;该质粒可在大肠杆菌中诱导表达hS100A2-GST融合蛋白

产率高;应用Glutathion-Sepharose4B球珠可获得纯度达92%的该蛋白。为后续有关hS100A2的研究打下了基础。

Abstract

Objective:To obtain human S100A2-GST fusion protein for further research on human S100A2(hS100A2)protein function and its interactions with other proteins and systems.Methods:hS100A2 gene from pHAHA-hS100A2 was subcloned into an expression vector pGST-moluc to construct pGST-moluc-hS100A2.The new plasmid was identified by digestion with XhoⅠand EcoRⅠ.The recombinant BL21 was induced with IPTG to express recombinant fusion protein hS100A2-GST

which was purified by Glutathion-Sepharose 4B ball beads

identified by SDS-PAGE and Western Blot

and quantified by Bradford method.Results:After the digestion

the recombinant plasmid was cutted into two fragments

300 bp and 5kb.After treated with IPTG

the recombinant BL21 strain expressed a protein

which was about 36 kD and was recognized specifically by an-ti-hS100A2.Its yield was 5 mg/L bacterial culture.After isolated by Glutathion-Sepharose 4B ball beads

its purity was 92%.Conclusion:hS100A2-GST expression plasmid pGST-moluc-hS100A2 was constructed successfully.hS100A2-GST fusion pro-tein could be expressed in Escherichia coli with higher yield and isolated with higher purity

which lays the foundation for the follow-up of the hS100A2 research.

关键词

人S100A2-GST融合蛋白诱导表达蛋白纯化蛋白鉴定

Keywords

HumanS100A2-GST fusion proteinInduced expressionprotein purificationProtein identification

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