摘要
目前激酶类蛋白的表达大多由昆虫细胞表达系统实现,采用BacMam系统来表达受体型酪氨酸激酶人FGFR1,旨在获得更接近天然结构与功能的重组蛋白。首先,将pDEST20载体的多角体蛋白启动子替换成CMV enhancer启动子,获得BacMam表达载体pDEST20-CMVe。通过Gateway系统,将FGFR1具有酪氨酸激酶活性的区域克隆至改造后的载体中。利用昆虫细胞Sf9产生病毒,P2代病毒转导哺乳动物细胞FreeStyle 293-F进行表达,经亲和纯化后,成功获得了75 kD的融合蛋白。通过磷酸化底物的反应检测此FGFR1激酶的活性。结果表明,BacMam系统是一个良好的表达重组激酶FGFR1的平台,并且所表达的激酶活性高于昆虫细胞表达的激酶蛋白。
The baculovirus expression system is one of the most popular methods for kinase proteins.Due to the fact that insect cells are not natural hosts for mammalian proteins,we used BacMam system to express the human FGFR1 for high yield and activity.After replacing PH promoter with CMV enhancer promoter on pDEST20 vector,the BacMam expression vector pDEST20-CMVe was constructed successfully.Then the intracellular region of FGFR1 as Tyr kinase was inserted under the control of CMV enhancer promoter by Gateway method.The FreeStyle 293-F cells were transduced with the BacMam viruses produced by Sf9 cells,and the recombinant protein expression was identified.Finally,the 75 kD fusion protein GST-FGFR1 was obtained after affinity purification.The result of phosphorylation reaction show that the activity of FGFR1 which expressed by BacMam system is higher than insect cell expression system.
出处
《生物技术通报》
CAS
CSCD
北大核心
2012年第10期114-118,共5页
Biotechnology Bulletin
