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| 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
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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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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.
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Received: 06 November 2009
Published: 29 April 2010
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Corresponding Authors:
Xian-Hui He
E-mail: thexh@jnu.edu.cn
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