Protein Purification and Kinase Assay Protocol

ARLPK1 and ARLPK2 were cloned into pMAL-C4X with N-terminal fusions to maltose-binding protein (MBP). MBP-ARLPK1, MBP-ARLPK2 and MBP-RIPK were induced with 0.3 mM isopropyl-β-D-thiogalactoside for 3 h at 28 °C and purified by amylose affinity chromatography from E. coli. The kinase domain of AKIK1 (547–892 aa) was cloned into pDEST-15 (Invitrogen) with an N-terminal fusion to glutathione S-transferase (GST). GST-AKIK1 was expressed in E. coli (BL21 strain) at 28 °C for 4 h and the recombinant protein purified with Glutathione Sepharose 4B (GE Healthcare). Kinase assays were performed using 3 μg of recombinant protein with [γ-³²P]-ATP. The assay was initiated by adding 1 ml (10 μCi) ³²P-ATP and incubated for 40 min at 30 °C. The reaction was terminated by the addition of 3 × laemmli loading buffer and subsequent incubation at 95 °C for 5 min. The proteins were separated on a 12% SDS–PAGE gel and signals were visualized by X-ray film exposure30 (link).