Pmal c2x

Recombinant PNG kinase complex was purified and the PNG kinase activity assay was performed as previously described (Hara et al., 2017 (link)) with modifications noted below. MBP-GNU was expressed and purified as described in Hara et al., 2017 (link). The gnu ORF was cloned into pMAL-c2x (NEB, Ipswich, MA). The gnu^P17L and gnu^ΔSAM mutant cDNAs were made using PCR and were cloned into pMAL-c2x (NEB, Ipswich, MA). The MBP-fused GNU^WT and mutants were expressed in BL21 Escherichia coli, purified following manufacturer protocols (NEB, Ipswich, MA), and dialyzed in TBS with 0.05% NP-40 and 1 mM DTT.
Recombinant PNG kinase complex (PNG-FLAG, PLU-His, GNU) containing 6 ng PNG-FLAG was incubated with 20 pg of purified MBP-GNU^WT, MBP-GNU^ΔSAM, or MBP-GNU^P17L (a negative control) together with 6 μg of Myelin Basic Protein (a PNG kinase in vitro substrate) in 10 μL PNG reaction buffer (20 mM Tris-HCl pH 7.5, 3 mM MnCl₂, 10 mM MgCl₂, 80 mM disodium β-glycerophosphate, 100 mM ATP, 1 mM DTT, and 1× complete EDTA-free protease inhibitor cocktail [Roche, Indianapolis, IN]) with 7.4 MBq/mL [γ-³²P]ATP at 30°C for 15 min. About 5 μL 3× LSB with 25 mM EDTA was added. The samples were heated at 96°C for 5 min and separated on 15% SDS-PAGE. After CBB staining, phosphorylated Myelin Basic Protein was detected by autoradiography.

Amylase resin, the plasmids pMAL-c2x and pTWIN1 are products of NEB, USA. Ni-NTA and proteases including factor Xa, enterokinase and thrombin are bought from Novagen, USA. E. coli strain BL21(DE3), and pET28b and pCDF-Duet1 vectors, and reagents for Western blot analysis are supplied by Novagen, USA. Ni-NTA superflow was obtained from Qiagen (Chatsworth, CA). Ultra-15 centrifugal filter tube equipped with Ultracel-10 membrane was supplied by Amicon (USA). The compounds including 2,4-dinitrophenylhydrazine (2,4-DNP), PLP and DL-DAP, PMSF, NEM, BzCl were from Sigma. Primer synthesis and DNA sequencing were performed by Invitrogen (Shanghai, China). Reagents used in plasmid construction and protein expression were from Takara (Dalian, China). The purified GST fused R3P is generously provided by Professor Zang Jianye, University of Science and Technology of China. The TEVp variant with five amino acid mutations are purified previously [23 (link)].

Full-length c-FLIP(s) and FADD were synthesized (Genscript, Piscataway, NJ, USA) and subcloned, respectively, into pET24b(+) (Novagen, Madison, WI, USA) and pMAL-C2X (New England Biolabs) expression vectors. The resulting constructs enabled the fusion of the corresponding protein with a C-terminal poly-histidine peptide, or a N-terminal Maltose Binding Protein (MBP).
All proteins were expressed in 1 mM IPTG-induced Rosetta-transformed bacterial cells with the expression vectors. After 18 h of induction at 37 °C, cells were harvested and pellets were resuspended in a lysis buffer (50 mM Tris-HCl, 100 mM NaCl, pH 8, 0.1% Triton), in addition to 0.1 mg/mL lysozyme and 1 mM PMSF (Phenylmethanesulfonyl fluoride), and incubated for 20 min at 4 °C. Cells were then lysed by freeze–thaw cycles, followed by sonification. Lysate was centrifuged at 34,000× g for 20 min at 4 °C, and supernatant was loaded on chromatographic media. HisPur^TM Ni-NTA Chromatography Cartridge 1 mL (Thermo Scientific, Waltham, MA, USA) or MBPTrap^TM HP 1 mL (GE Healthcare, Chicago, IL, USA) were, respectively, used for purification of c-FLIP(s) or FADD, following the column’s manufacturer procedures. Protein purity was assessed by SDS-PAGE analysis and concentrations were qualified using the Bio-Rad protein assay, based on the Bradford dye-binding method.