| KDR酪氨酸激酶的克隆表达及纯化 |
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| 引用本文： | 刘春平,张洋,李元. KDR酪氨酸激酶的克隆表达及纯化[J]. 生物工程学报, 2008, 24(9): 1545-1549 |
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| 作者姓名： | 刘春平 张洋 李元 |
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| 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,卫生部抗生素生物工程重点实验室,北京,100050 |
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| 基金项目： | 国家高技术研究发展计划(863计划) |
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| 摘 要： | VEGF作为一种血管内皮细胞有丝分裂原,通过与内皮细胞表面特定受体结合,从而导致新生血管的形成,其中VEGFR-2(KDR/FIk-1)在肿瘤血管形成中起重要作用,因此其抑制剂有可能发展成为治疗肿瘤的新药.采用大肠杆菌成功表达了具有酪氨酸激酶活性的KDR酪氨酸激酶催化域(KDR-CD).采用RT-PCR从人脐静脉内皮细胞中提取总RNA,获得KDR-CD的编码基因,将其克隆至pET-30a载体,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,Western blotting结果显示表迭的KDR-CD蛋白自身磷酸化,重组KDR-CD蛋白具有利用ATP催化底物发生磷酸化反应的激酶活性.
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| 关 键 词： | 血管生成 KDR酪氨酸激酶 大肠杆菌 酪氨酸激酶活性 |
| 收稿时间： | 2008-01-14 |
Cloning, Expression and Purification of KDR Tyrosine Kinase |
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| Chunping Liu,Yang Zhang and Yuan Li. Cloning, Expression and Purification of KDR Tyrosine Kinase[J]. Chinese journal of biotechnology, 2008, 24(9): 1545-1549 |
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| Authors: | Chunping Liu Yang Zhang Yuan Li |
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| Affiliation: | Key Laboratory of Biotechnology of Antibiotics, Ministry of Health, Institute of Medicinal Biotechnology; Chinese Academy of Medical Science and Perking Union Medical College, Beijing 100050, China;Key Laboratory of Biotechnology of Antibiotics, Ministry of Health, Institute of Medicinal Biotechnology; Chinese Academy of Medical Science and Perking Union Medical College, Beijing 100050, China;Key Laboratory of Biotechnology of Antibiotics, Ministry of Health, Institute of Medicinal Biotechnology; Chinese Academy of Medical Science and Perking Union Medical College, Beijing 100050, China |
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| Abstract: | The catalytic domain of KDR kinase (KDR-CD) was amplified from RNA of HUVCEs cells with RT-PCR and expressed in E. coli BL21(DE3) by plasmid pET30a as vector. The recombinant protein was purified with affinity chromatography (Ni-NTA). Western blotting showed that the recombinant KDR-CD was phosphorylated in E. coli BL21(DE3). The recombinant KDR-CD was identified to have kinase activity catalyzing the substrate phosphorylated with ATP in the enzymatic reaction. |
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| Keywords: | angiogenesis KDR tyrosine kinase E. coli kinase activity. |
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