Expression and purification of active receptor interacting protein 1 kinase using a baculovirus system

Abstract

Receptor Interacting Protein 1 (RIP1) kinase is one of the key mediators of tumor necrosis factor alpha (TNF-α) 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, 1-3mg of highly purified RIP1 kinase was typically obtained from a 1L 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.

Figure 1

Expression of active RIP1-His. A. Coomassie blue stained 12% SDS-PAGE gel of different baculovirus infected samples purified using His-select beads and eluted with 500 mM imidazole: M – molecular weight marker, 1 – cells alone, 2 – Cdc37 infected cells, 3 – RIP1-His infected cells, 4 – Cdc37 and RIP1-His infected cells. B. Structures of the different necrostatins used in this study. C. ³²P autophosphorylation kinase reactions from the eluents of RIP1-His infection (no Cdc37) and Cdc37 and RIP1-His co-infection, treated with either DMSO (D) control or 30 μM of optimized RIP1 inhibitor Nec-1 (N1). RIP1-His expressed alone was not inhibited by Opt Nec-1.

Figure 2

Optimization of RIP1-His co-baculovirus infection with Cdc37. A. The expression of RIP1-His was tested by mixing RIP1-His baculovirus with increasing concentrations of Cdc37 baculovirus. The expression of RIP1-His was detected by western blot using a RIP1 antibody. The lanes are labeled according to the MOI of Cdc37. Tubulin was used to normalize the amount of sample in each lane. B. Time-course of infection was performed by taking samples every 12 hours after co-baculovirus infection using MOIs of 0.034 for RIP1-His and 0.0064 for Cdc37. The expression of RIP1-His was monitored by western blot. At 72 hours post-infection, RIP1-His starts to get modified and degraded so 60 hours was determined to be optimal.

Figure 3

Purification and activity of RIP1-His. A. RIP1-His and Cdc37 baculoviruses were coinfected into 1L of Sf9 cells at a density of 3×10⁶ cells/ml. Sixty hours post-infections, the cells were lysed as described in the materials and methods. The supernatant was loaded onto a 3 ml His-select column. Coomassie blue stained 12% SDS-PAGE gels represent the different imidazole steps from the His-select column: M – molecular weight marker, lanes 1-2 flow through from loading, lanes 3-5 0 mM imidazole step, lanes 6-8 10 mM imidazole step, lanes 9-18 50 mM imidazole step and lanes 19-28 500 mM imidazole step. B. Chromatogram of the absorbance at 280 nm of the 50 mM imidazole eluted fractions from A injected onto a Superdex 200 SEC column (GE Healthcare). C. Coomassie blue stained 12% SDS-PAGE gel of Superdex 200 SEC elution volume collected in 1 ml fractions. D. Elution volume fractions from B were tested in the radiometric kinase assay with protein concentration of 1 μM with either DMSO (D) or 15 μM opt Nec-1 (N1). E. The final purified RIP1-His protein at 2 μM was tested in the radiometric kinase assay with the various necrostatins at 30 μM (Figure 1B). RIP1-His is inhibited by necrostatins but not by the inactive analogs.

Figure 4

Thermal shift assay with RIP1-His A. RIP1-His at 9 μM in the presence of 3% DMSO or 18 μM inhibitors was subjected to thermal denaturation using Sypro Orange as a fluorescent tracer. Each sample was run in duplicate and one representative thermal trace is shown. B. The T_m for RIP1-His with and without inhibitors was calculated as described in the materials and methods. The shift in T_m from RIP1-His DMSO to RIP1-His inhibitors demonstrates that ori Nec-1, opt Nec-1, and Nec-3 increased RIP1-His T_m, while Nec-1i and Nec-3i have a minimal effect on RIP1-His T_m demonstrating that they do not bind.