毛白杨PtoeIF5A4的原核表达、纯化及鉴定

生物技术通报 ›› 2014, Vol. 0 ›› Issue (5): 83-87.

毛白杨PtoeIF5A4的原核表达、纯化及鉴定

张杰伟管阳朱丹陈亚娟丁莉萍王宏芝魏建华

(北京市农林科学院北京农业生物技术研究中心农业基因资源与生物技术北京市重点实验室，北京 100097)

收稿日期:2013-10-09 出版日期:2014-05-23 发布日期:2014-05-24
作者简介:张杰伟，男，助理研究员，研究方向：园林分子生物学；E-mail：jwzhang919@163.com
基金资助:
国家自然科学基金项目(30972392)，国家高技术研究发展计划("863"计划)项目(2011AA100201)，国家重点基础研究发展计划("973"计划)项目(2012CB114501)

Expression, Purification and Characterization of Populus tomentosa PtoeIF5A4 in Prokaryote

Zhang Jiewei Guan Yang Zhu Dan Chen Yajuan Ding Liping Wang Hongzhi Wei Jianhua

(Beijing Agro-Biotechnology Research Center，Beijing Key Laboratory of Agricultural Genetic Resources and Biotechnology，Beijing Academy of Agriculture and Forestry Sciences，Beijing 100097)

Received:2013-10-09 Published:2014-05-23 Online:2014-05-24

摘要/Abstract

摘要： 为获取大量高纯度的毛白杨PtoeIF5A4蛋白以便研究其特性和生物学功能，以pGEM-T Easy-PtoeIF5A4质粒为模板，PCR扩增毛白杨PtoeIF5A4基因的cDNA编码区，测序正确后，经酶切将其插入到原核表达载体pET28a上，然后将表达载体转入大肠杆菌表达菌株BL21(DE3)，经不同浓度IPTG诱导培养后发现，0.1 mmol/L IPTG，28℃诱导4 h时目的蛋白最多，且该重组蛋白主要以可溶性的形式存在。采用亲和纯化从可溶性蛋白中纯化到大小为18 kD的蛋白条带。Western blotting分析发现亲和纯化所得蛋白可以与His单克隆抗体发生免疫交叉反应，表明纯化所得蛋白是PtoeIF5A4重组蛋白。

关键词: 毛白杨, PtoeIF5A4, 原核表达, 蛋白纯化

Abstract: To obtain the PtoeIF5A4 protein of poplar with high purity in a large scale, its full length CDS was amplified by PCR using pGEM-T Easy-PtoeIF5A4 as template and verified by sequencing, and then was inserted into pET28a vector containing histidine. The recombinant pET28-PtoeIF5A4 plasmid was obtained and transformed into E. coli BL21(DE3). The recombinant fusion protein was produced by culturing the transformed E. coli BL21(DE3)with subsequent IPTG induction, and was observed predominantly in the supernatant of cellular extracts when cells were cultured at 28℃ and induced with 0.1 mmol/L IPTG for 4 h. The fusion protein was purified from the soluble fraction using a column of Ni²⁺ Chelating Sepharose Fast Flow, and proofed molecular mass was 18 kD. The anti-His monoclonal antibody recognized the protein, which indicated that it was PtoeIF5A4 recombinant protein.

Key words: Populus tomentosa, PtoeIF5A4, Prokaryotic expression, Protein, Purification

张杰伟, 管阳, 朱丹, 陈亚娟, 丁莉萍, 王宏芝, 魏建华. 毛白杨PtoeIF5A4的原核表达、纯化及鉴定[J]. 生物技术通报, 2014, 0(5): 83-87.

Zhang Jiewei, Guan Yang, Zhu Dan, Chen Yajuan, Ding Liping, Wang Hongzhi, Wei Jianhua. Expression, Purification and Characterization of Populus tomentosa PtoeIF5A4 in Prokaryote[J]. Biotechnology Bulletin, 2014, 0(5): 83-87.

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