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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2010, Vol. 30 Issue (04): 39-43    
研究报告     
重组人ASCT2的C-末端胞外结构域的原核表达、纯化及鉴定
欧阳东云1,2,徐丽慧3,高琦1,2,郭贺1,2,陈清1,2,何贤辉1,2**
1.暨南大学组织移植与免疫实验中心 广州 510632
2.暨南大学再生医学教育部重点实验室 广州 510632
3.暨南大学生物工程研究所 广州 510632
Expression, Purification and Identification of Human Recombinant ASCT2 C-Terminal Extracellular Domain in Escherichia coli
OUYANG Dong-yun1,2,XU Li-hui3,GAO Qi1,2,GUO He1,2,CHEN Qing1,2,HE Xian-hui1,2
1.Institute of Tissue Transplantation and ImmunologyJinan University,Guangzhou 510632,China
2.Key Laboratory for Regenerative Medicine of Ministry of Education, Jinan University, Guanzhou 510632,China
3.Institute of Bioengineering, Jinan University, Guangzhou 510632, China
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摘要：

中性氨基酸转运蛋白（ASCT2）是人类内源性病毒的包膜蛋白合胞素在细胞膜上的主要受体，其最大的胞外结构域存在于C-末端的105个氨基酸（以下简称TAIL）。通过RT-PCR方法从人乳腺癌MCF-7细胞中克隆ASCT2基因编码区全长序列，再从中扩增ASCT2的TAIL序列，与pET-41b（克隆位点的N-和C-端分别有谷胱甘肽转移酶和His6标签序列）连接构建原核表达载体，重组质粒在大肠杆菌中获得表达，免疫印迹显示重组蛋白TAIL在细菌裂解液上清和沉淀（包涵体）中均有表达，可溶性部分经亲和层析纯化获得高纯度的TAIL蛋白，该蛋白可结合在表达合胞素的MCF-7细胞表面，提示其可能具有结合合胞素的活性。
关键词： 合胞素;  中性氨基酸转运蛋白;  胞外结构域;  原核表达    
Abstract:

The C-terminal (comprising 105 amino acids) of neutral amino acid transporter ASCT2 (named as TAIL in this study) is the largest extracellular domain of the protein. ASCT2 is reported to be the main receptor for the envelope of human endogenous retrovirus W family (syncytin) on cell membrane. In this study, ASCT2 cDNA was derived from human breast cancer cell line MCF-7 by RT-PCR method. DNA fragment encoding the TAIL peptide was inserted into pET-41b vector and the recombinant plasmid was expressed in Escherichia coli (E. coli). TAIL peptide, which was recombinated with a glutathione S-transferase (GST) at the N-terminal and a His6 tag at the C-terminal, was expressed in E. coli cells. SDS-PAGE and Western blotting analysis demonstrated that the recombinant protein existed both in the supernatant and the pellet part of E. coli lysates. The supernatant was further purified by affinity chromatography. Highly pure recombinant protein could bind with MCF-7 cells which were reported to express syncytin molecules on their membranes. These results suggest that the recombinant protein has the potential to bind with syncytin molecules.
Key words: Syncytin    Neutral amino acid transporter type 2    Extracellular domain    Prokaryotic expression
收稿日期: 2009-11-06 出版日期: 2010-04-29
基金资助:

广东省自然科学基金资助项目（8451063201000340）
通讯作者: 何贤辉     E-mail: thexh@jnu.edu.cn
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欧阳东云
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引用本文:

欧阳东云 徐丽慧 高琦 郭贺 陈清 何贤辉. 重组人ASCT2的C-末端胞外结构域的原核表达、纯化及鉴定[J]. 中国生物工程杂志, 2010, 30(04): 39-43.

OU Yang-Dong-Yun, XU Li-Hui, GAO Qi, GUO He, CHEN Qing, HE Xian-Hui. Expression, Purification and Identification of Human Recombinant ASCT2 C-Terminal Extracellular Domain in Escherichia coli. China Biotechnology, 2010, 30(04): 39-43.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/    或    https://manu60.magtech.com.cn/biotech/CN/Y2010/V30/I04/39

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