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人EGF受体L2结构域在大肠杆菌中表达、包涵体柱上复性及纯化
引用本文:徐丽慧,洪岸,何贤辉.人EGF受体L2结构域在大肠杆菌中表达、包涵体柱上复性及纯化[J].生物工程学报,2005,21(4):597-603.
作者姓名:徐丽慧  洪岸  何贤辉
作者单位:1. 暨南大学组织移植与免疫教育部重点实验室,广州,510632;暨南大学生物工程研究所,广州,510632;广东省生物工程药物重点实验室,广州,510632;教育部基因组药物工程研究中心,广州,510632
2. 暨南大学生物工程研究所,广州,510632;广东省生物工程药物重点实验室,广州,510632;教育部基因组药物工程研究中心,广州,510632
3. 暨南大学组织移植与免疫教育部重点实验室,广州,510632
基金项目:国家自然科学基金重点(No.30230350和No.30371651),国家“十五”重大专项基金(No.2002AA2Z3344),广东省“十五”重大专项基金(No.2001A1090208)资助项目~~
摘    要:以PCR方法从克隆的EGFR胞外区cDNA中扩增编码EGFR-L2结构域的DNA片段,在其3′端加入编码His6标签的序列,与pET-3c连接构建EGFR-L2原核表达载体。该蛋白在大肠杆菌BL21(DE3)中获得高效表达,免疫印迹分析表明表达产物全部以包涵体形式存在,分步透析法和稀释法都不能获得可溶性复性产物,而Ni2+-NTA柱上复性法不仅能够获得可溶性的EGFR-L2蛋白,而且产物同时得到高度纯化,纯度>95%,复性的EGFR-L2与其配基EGF具有特异性的结合活性,但亲和力较低。这表明His6标签不但便于纯化目标蛋白,而且可利用Ni2+-NTA柱进行柱上复性,适用于不易通过常规方法复性的重组蛋白的制备。

关 键 词:表皮生长因子受体,L2结构域,原核表达,包涵体,重折叠
文章编号:1000-3061(2005)04-0597-07
修稿时间:2005年1月31日

On-column Refolding and Purification of Human EGF Receptor L2 Domain Inclusion Body Overexpressed in Escherichia coli
XU Li-hui,HONG An,HE Xian-hui.On-column Refolding and Purification of Human EGF Receptor L2 Domain Inclusion Body Overexpressed in Escherichia coli[J].Chinese Journal of Biotechnology,2005,21(4):597-603.
Authors:XU Li-hui  HONG An  HE Xian-hui
Institution:Key Laboratory of Ministry of Education for Tissue Transplantation and Immunology, Jinan University, Guangzhou 510632, China.
Abstract:The human epidermal growth factor receptor (EGFR) extracellular region (residues 1-621) consists of four subdomains, i.e. L1, S1, L2, and S2.The L2 domain (EGFR-L2) is composed of residues 311-479 and plays a major role in ligand-binding. Due to the high content of cysteine residues (42 cysteines) in the S1 and S2 domains, it is quite difficulty to get a correctly refolded product of the complete EGFR extracellular domain. In contrast, only 4 cysteine residues are present in EGFR-L2 domain. The aim of the present study is to prepare a soluble EGFR-L2 domain from the recombinant protein inclusion body overexpressed in Escherichia coli (E. coli). DNA fragment encoding EGFR-L2 containing a polyhistidine-tag at the carboxyl terminus was amplified by PCR from the cDNA of EGFR extracellular region, and was inserted into pET-3c to construct the prokaryotic expression vector. The target protein was highly expressed in E. coli BL21(DE3) strain and was only present in the inclusion body as revealed by immunoblotting analysis. No soluble product could be refolded through dilution or stepwise dialysis strategies. However, on-column refolding of denatured EGFR-L2 bound to Ni~ 2+-NTA produced a soluble one. Furthermore,the soluble EGFR-L2 was simultaneously purified to high purity (>95%) through eluting from the same Ni~ 2+-NTA column with a linear imidazole gradient. The refolded EGFR-L2 had specific binding activity with the cognate ligand EGF, although its affinity was low. These results suggest that a polyhistidine-tag fused with a recombinant protein facilitate not only the purification but also the renaturation of the target product through on-column refolding. Besides, this refolding strategy may be suitable for the preparation of those recombinant proteins which are hard to refold through conventional approaches.
Keywords:epidermal growth factor receptor  L2 domain  prokaryotic expression  inclusion body  refolding
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