Liu Chunping, Zhang Yang, Li Yuan
Key Laboratory of Biotechnology of Antibiotics, Ministry of Health, Institute of Medicinal Biotechnology, Beijing 100050, China.
Sheng Wu Gong Cheng Xue Bao. 2008 Sep;24(9):1545-9.
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.
通过逆转录聚合酶链反应（RT-PCR）从人脐静脉内皮细胞（HUVCEs）的RNA中扩增出KDR激酶的催化结构域（KDR-CD），并以质粒pET30a为载体在大肠杆菌BL21(DE3)中表达。重组蛋白通过亲和层析（镍-氮三乙酸）进行纯化。蛋白质免疫印迹法显示重组KDR-CD在大肠杆菌BL21(DE3)中发生了磷酸化。经鉴定，重组KDR-CD在酶促反应中具有催化底物被ATP磷酸化的激酶活性。
