Recombinant protein expression

The pDEST17 vector (Invitrogen) containing the gene of interest was used to express recombinant proteins (IRAK3, IRAK3G361L and IRAK3R372L) in BL21-AI E. coli cells (Invitrogen, USA) as previously described⁶³. The BL21-AI E. coli system is designed for tight regulation and expression of toxic proteins from T7 promoter based systems under the control of the arabinose inducible araBAD promoter^64–66. High expressing cultures were selected and were upscaled to 500 ml liquid cultures grown to an OD600 of ~0.4 before induction with 0.2% L-arabinose (Sigma-Aldrich, Australia) and grown for a further 3 hours at 20 °C. Cells were harvested by centrifugation. Proteins were purified by affinity chromatography using the Ni-NTA agarose beads (QIAGEN, Germany) under native conditions following protocol 12 of the QIAexpressionist manual (QIAGEN, Germany) in the presence of 30 mM imidazole to decrease the unspecific binding onto the Ni-NTA beads and in the presence of complete EDTA-free protease inhibitor cocktail tablets (Roche, Australia). Eluted protein was concentrated using Vivaspin® 20 centrifugal concentrators (Sartorius Stedim Biotech, Germany) with a molecular weight cut-off of 30 kDa in the presence of 1 mM phenylmethylsulfonyl fluoride (PMSF) and protein was quantitated using a Nanodrop® ND-1000 Spectrophotometer protein at A₂₈₀. Proteins were separated by SDS-PAGE using 16% separating and 4% stacking gels and run at 200 volts for 50 minutes (BioRAD mini electrophoresis setup), stained with Coomassie blue (0.03% (w/v) Coomassie Brilliant blue, 50% methanol, 10% glacial acetic acid and 40% distilled water), and destained with destain solution (40% methanol, 10% acetic acid and 50% distilled water).