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Cloning and Expression of EGFR Tyrosine Kinase and Construction of the Screening Model for the Enzymatic Inhibitors
作者姓名:张洋  刘春平  李元
作者单位:中国医学科学院中国协和医科大学医药生物技术研究所 中国医学科学院中国协和医科大学医药生物技术研究所 中国医学科学院中国协和医科大学医药生物技术研究所
摘    要:表皮生长因子受体(Epidermal growth factor receptor,EGFR)属受体酪氨酸激酶(Tyrosine kinase,TK)家族,其胞内的酪氨酸激酶在细胞信号转导通路中具有十分重要的作用。许多肿瘤的发生、发展都与EGFR胞内酪氨酸激酶的异常表达密切相关。因此,EGFR胞内酪氨酸激酶的抑制剂有可能成为治疗肿瘤的有效药物。本研究从人脐静脉内皮细胞(HUVEC) 提取总RNA,采用RT-PCR获得EGFR酪氨酸激酶催化域的编码基因。将其克隆至载体pET-30a,在E.coli BL21(DE3)中进行了成功表达,采用Ni-NTA亲和层析对其进行了纯化。通过对酶的活性的测定,证明重组EGFR酪氨酸激酶蛋白具有利用ATP催化底物发生磷酸化反应的激酶活性。以该重组激酶为靶位构建了酶抑制剂筛选模型,拟对微生物代谢产物进行筛选。

关 键 词:EGFR酪氨酸激酶  克隆表达  重组蛋白  抑制剂筛选模型  
收稿时间:2008-06-24
修稿时间:2008-08-04

Cloning and Expression of EGFR Tyrosine Kinase and Construction of the Screening Model for the Enzymatic Inhibitors
ZHANG Yang,LIU Chun-ping,LI Yuan.Cloning and Expression of EGFR Tyrosine Kinase and Construction of the Screening Model for the Enzymatic Inhibitors[J].China Biotechnology,2008,28(10):12-17.
Authors:ZHANG Yang  LIU Chun-ping  LI Yuan
Abstract:As a member of tyrosine kinases (TK) family, Epidermal growth factor receptor(EGFR) has an activity of intrinsic protein tyrosine kinase, which plays an essential role in the regulation of signal transduction in the cells. Due to the abnormal expression of EGFR-TK, the certain type cancers may developed and progressed. Based on that, the inhibitors of EGFR-TK could be effective medicines for the treatment of cancer. In this study, the EGFR-TK domain was amplified by RT-PCR with RNA of HUVCEs cells as the template and expressed in E.coli BL21(DE3) using plasmid pET30a as vector. The recombinant protein was purified with the affinity chromatography (Ni-NTA), which was identified to have kinase activity catalyzing the substrate phosphorylated with ATP in the enzymatic reaction. Using the recombinant EGFR-TK as target, the screening model for enzymatic inhibitors was constructed.
Keywords:EGFR tyrosine kinase  Cloning and expresssion  Recombinant protein  The screening model for inhibitors
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