重组人ASCT2胞外结构域ECL2在大肠杆菌中的表达及其鉴定

Expression and Identification of Human Recombinant ASCT2 Extracellular Domain ECL2 in Escherichia coli

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,ECL2是该受体的其中一个较大的胞外结构域.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的胞外区ECL2序列,与pET-41b连接构建原核表达载体,重组质粒在大肠杆菌中获得高效表达,重组蛋白在N-和C-端分别融合谷胱甘肽转移酶(GST)和His6标签,融合蛋白在上清液和包涵体中均有表达,可溶性部分经亲和层析纯化获得高纯度的重组蛋白,该蛋白可结合在表达合胞素的MCF-7细胞表面,具有结合合胞素的潜在活性,这些结果为进一步研究ASCT2与合胞素的相互作用奠定了基础.

The neutral amino acid transporter ASCT2 is reported to be the main receptor for the envelope of human endogenous retrovirus W family（named as syncytin） on cell membrane.And ECL2 is one of the ASCT2 extracellular domains,covering the amino acid 153 ~227 sequence.Here,ASCT2 cDNA was amplified by RT-PCR method from the total RNA extract of human breast cancer cell line MCF-7.DNA fragment encoding the ECL2 peptide was inserted into pET-41b vector and the recombinant prokaryotic expression plasmid was highly expressed in Escherichia coli（E.coli）.The recombinant protein,which contained a glutathione S-transferase（GST） and a His6 tag at the N-and C-terminal of ECL2 peptide respectively,was present both in the supernatant and the pellet part of E.coli lysates.The supernatant was further purified by affinity chromatography.High pure recombinant protein could bind with MCF-7 cells which were reported to express syncytin molecules on their membranes.These results demonstrated that the recombinant protein has the potential to bind with syncytin molecules,which paved the way for further research on interaction between ASCT2 and syncytin.