Tartaglia Lawrence J, Bennett Antonette, Plattner Alexander S, Muzyczka Nicholas, Ling Chen, Srivastava Arun, Agbandje-McKenna Mavis
Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, FL 32610, USA.
Protein Expr Purif. 2013 Nov;92(1):21-8. doi: 10.1016/j.pep.2013.08.013. Epub 2013 Aug 29.
The α5β1 integrin heterodimer is involved in many cellular processes and is an anti-cancer therapeutic target. Therefore, access to quantities of protein suitable for studies aimed at understanding its biological functions is important. To this end, a large-scale protein expression system, utilizing the recombinant baculovirus/SF9 insect cell expression system, was created to produce the extracellular domain of the α5β1 integrin. An incorporated 8X-histidine tag enabled one-step nickel-column purification. Following sequence confirmation by LC-MS/MS, the conformation of the heterodimer was characterized by native dot blot and negative stain electron microscopy. Cellular transduction inhibition studies confirmed biological activity. The system allows expression and purification of α5β1 integrin in quantities suitable for an array of different experiments including structural biology.
α5β1整合素异二聚体参与许多细胞过程，是一个抗癌治疗靶点。因此，获得适合用于旨在了解其生物学功能研究的大量蛋白质很重要。为此，利用重组杆状病毒/SF9昆虫细胞表达系统创建了一个大规模蛋白质表达系统，以生产α5β1整合素的胞外结构域。引入的8X组氨酸标签实现了一步镍柱纯化。通过LC-MS/MS进行序列确认后，通过天然点杂交和负染电子显微镜对异二聚体的构象进行了表征。细胞转导抑制研究证实了其生物活性。该系统能够表达和纯化适合一系列不同实验（包括结构生物学实验）的α5β1整合素。
