人源EFA6A蛋白Sec7结构域的克隆表达与纯化

生物技术通报 ›› 2013, Vol. 0 ›› Issue (5): 81-85.

人源EFA6A蛋白Sec7结构域的克隆表达与纯化

谢长林¹, 芮斌¹, 江娜¹, 赵晨², 唐雅珺², 文汉¹

(1.安徽农业大学生命科学学院，合肥 230036；2.中国科学技术大学生命科学学院，合肥 230026)

收稿日期:2013-01-29 修回日期:2013-05-24 出版日期:2013-05-24 发布日期:2013-05-24
作者简介:谢长林，男，硕士，研究方向：蛋白质结构与功能；E-mail：xclkush1988@163.com；芮斌同为第一作者

Cloning，Expression，Purification of Sec7 Domain of hEFA6A Protein

Xie Changlin¹, Rui Bin¹, Jiang Na¹, Zhao Chen², Tang Yajun², Wen Han¹

(1. College of Life Science of Anhui Agriculture University, Hefei 230036；2. College of Life Science of University of Science & Technology China, Hefei 230026)

Received:2013-01-29 Revised:2013-05-24 Published:2013-05-24 Online:2013-05-24
About author:唐雅珺，女，博士，讲师，研究方向：蛋白质结构与功能；E-mail：tangyj@ustc.edu.cn文汉，男，硕士，副教授，研究方向：生物大分子提取；E-mail：lifwen1000@163.com

摘要/Abstract

摘要： 作为小GTP酶Arf6的鸟甘酸交换因子(GEF)，人EFA6A蛋白主要包含PH和Sec7两个结构域，Sec7是行使GEF功能的核心区域。通过分析Jpred、Uniprot等生物信息学软件的预测结果，从全长1 024 aa中选取的重组Sec7结构域的边界为506-719，共214 aa。以人脑cDNA文库为模板，通过优化PCR程序成功扩增出Sec7基因，经Nde I和Xho I双酶切后亚克隆至原核表达载体p28a中，成功构建p28-Sec7重组子，测序结果与NCBI中公布的序列100%吻合。将重组质粒p28- Sec7转化至BL21-Gold(DE3)宿主菌中，终浓度0.3 mmol/L IPTG、16℃、24 h诱导表达，重组蛋白经过Ni柱和分子筛两步纯化。试验结果显示，重组Sec7成功表达，性质均一，纯度高于95%，表达量为70 mg/L。

关键词: Arf6, 人EFA6A, Sec7, 表达, Ni-NTA柱, 分子筛

Abstract: As a GEF for the small GTPase Arf6, hEFA6A contains two domains which are pH domain and Sec7 domain. Sec7 domain is the key central domain that executes the function as a GEF. By analyzing the prediction results from Jpred and Uniprot, we chose the boundary contained 214 aa (506-719) from the full-length (1 024 aa) of hEFA6A. Sec7 gene fragment was cloned from homo brain cDNA library and ligated to p28a vector after Nde I and Xho I double digestion. Then the constructive vector p28a-Sec7 was transferred to BL21-Gold (DE3) competent cell for expression. The final concentration of IPTG was 0.3 mmol/L and E.coli cells were cultivated at 16℃ for 24 hours. The recombinant protein was then purified by Ni-NTA affinity chromatography and GE superdexS200 gel filtration column. Our results showed that Sec7 gene and the constructive vector p28a-Sec7 were acquired successfully. The sequence of Sec7 was completely matched with the sequence reported in NCBI. We had a high expression of 70 mg/L and harvested the 95% purity target protein successfully.

Key words: Arf6 hEFA6A, Sec7, Expression, Ni-NTA column, Gel filtration

谢长林, 芮斌, 江娜, 赵晨, 唐雅珺, 文汉. 人源EFA6A蛋白Sec7结构域的克隆表达与纯化[J]. 生物技术通报, 2013, 0(5): 81-85.

Xie Changlin, Rui Bin, Jiang Na, Zhao Chen, Tang Yajun, Wen Han. Cloning，Expression，Purification of Sec7 Domain of hEFA6A Protein[J]. Biotechnology Bulletin, 2013, 0(5): 81-85.

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