Expression and purification of active human receptor interacting protein 1 kinase using a baculovirus system
Date
2014-06
Journal Title
Journal ISSN
Volume Title
Publisher
Protein Expression and Purification
Abstract
Receptor Interacting Protein 1 (RIP1) kinase is one of the key mediators of tumor necrosis factor alpha (TNF-?) signaling and is critical for activation of necroptotic cell death. We developed a method for expression of recombinant kinase, utilizing baculovirus co-infection of Cdc37, an Hsp90 co-chaperone, and RIP1-His, followed by a two-step purification scheme. After optimization, 1–3 mg of highly purified RIP1 kinase was typically obtained from a 1 L of Sf9 cells. The recombinant protein displayed kinase activity that was blocked by RIP1 inhibitors, necrostatins. The purified protein was used to develop a simple and robust thermal shift assay for further assessment of RIP1 inhibitors.
Description
Keywords
RIP1 kinase, Cdc37, Necrostatins, Baculovirus, Thermal shift assay
Citation
Copyright 2013 Protein Expression and Purification. This is a post-print version of a published paper that is available at: https://www.sciencedirect.com/science/article/pii/S1046592813000478. Recommended citation: Maki, Jenny L., J. Tres Brazell, Xin Teng, Gregory D. Cuny, and Alexei Degterev. "Expression and purification of active receptor interacting protein 1 kinase using a baculovirus system." Protein expression and purification 89, no. 2 (2013): 156-161.doi: 10.1016/j.pep.2013.03.002. This item has been deposited in accordance with publisher copyright and licensing terms and with the author's permission.