HER-2/neu胞外配体结合区2在大肠杆菌中可溶性表达及纯化

Soluble Expression and Purification of HER-2/neu Extracellular Receptor Ligand Domain 2

用PCR技术扩增HER 2 neu胞外配体结合区 2 (RLD2 )cDNA ,并将扩增的基因片段克隆于硫氧还蛋白 (TrxA)原核表达载体中 ,获得TrxA RLD2融合蛋白的可溶性表达 .通过插入偶联翻译序列 ,实现TrxA与RLD2蛋白在大肠杆菌中的共表达 .表达产物经免疫印记检测可被抗HER 2 neu特异性抗体识别 .经离子交换层析和钴亲和层析纯化 ,RLD2蛋白的纯度达 90 % .用质谱法分析RLD2蛋白的分子量 ,与预期值相符 .结果表明 ,利用TrxA表达体系在大肠杆菌中获得了HER 2

HER 2/neu receptor ligand domain 2 (RLD2) cDNA was amplified by PCR, cloned into the prokaryotic expression vector containing thioredoxin A (TrxA) gene and expressed in soluble form as TrxA RLD2 fusion protein. TrxA and RLD2 was co expressed by inserting a tandem sequence of stop and start codon into the recombinant plasmid. Western blotting showed that the expressed protein could react with the specific antibody against proto oncoprotein HER 2/neu. With the application of DEAE Sepharose FF and cobalt based affinity resin, the purity of RLD2 obtained was over 90%. The purified protein was further subject to mass spectroscopy molecular weight monitoring, and the molecular weight of RLD2 protein was identical to that expected. By using TrxA expression system, the soluble expression of RLD2 protein was obtained with high efficiency.