Complete protease inhibitor cocktail

In vitro kinase assay was done, as shown in the former studies [50 ]. Briefly, GST protein expressed in Escherichia coli was lysed with a RIPA buffer (50-mM Tris-HCl pH 7.4, 150-mM NaCl, 0.1% SDS, 0.5% DOC, 1% NP-40) having a complete protease inhibitor cocktail (Roche) and bound to Glutathione Sepharose 4B beads (GE Healthcare). Then, the GST protein was rinsed with a kinase buffer (40-mM Tris-HCl pH 7.4, 20-mM MgCl₂, 0.1-mg/ml bovine serum albumin, and 50-μM dithiothreitol) and eluted using a kinase buffer containing 20-mM reduced glutathione. The RC-DC Protein Assay (Bio-Rad) was applied to measure the GST protein levels.
For the kinase assay, 16.7-ng/μl substrate candidate protein, 0.33-ng/μl ARK5 kinase, 33.3-nCi/μl (11 nM) [γ32-P] ATP, and 50-µM non-radiolabeled ATP were mixed in a kinase buffer (reaction volume of 30 μl) and stored for 30 min at 30 °C. The end of the reaction was marked by blending with 4× Laemmli sample buffer (40% glycerol, 270-mM Tris-HCl pH 6.8, 8% SDS, 20% 2-mercaptoethanol, 0.006% BPB). Then, the samples were detached by SDS-PAGE. In the end, the autoradiography image was obtained with BAS-5000 (GE Healthcare), Image Reader BAS-5000 Version 1.8 (Fujifilm), and Multi Gauge Version 3.1 (Fujifilm) software.

sNASP was expressed in bacteria as an N-terminal (His)₆ fusion construct and was purified over Nickel-NTA beads (GE Healthcare) in 300 mM sodium chloride, 50 mM Tris–HCl pH 8.0, 0.1% NP40, 20 mM imidazole and Complete protease inhibitor cocktail (Roche). Protein was eluted in the same buffer with 250 mM imidazole, but without protease inhibitors. The (His)₆ tag was cleaved overnight by TEV protease, whilst concomitantly dialysing to 100 mM sodium chloride, 20 mM Tris–HCl pH 8.0, 1 mM EDTA and 5 mM dithiothreitol. Cleaved protein was further purified by ion-exchange using a ResourceQ column (GE Healthcare) before gel filtration chromatography in 200 mM sodium chloride, 20 mM Hepes-KOH pH 7.6 and 1 mM EDTA using a 26/60 Superdex 200 column (GE Healthcare). The purified protein was concentrated and stored in aliquots at −80°C. Homologues, truncations and point mutants of sNASP were expressed and purified using the same method. MBP-H3 peptide fusions were expressed in bacteria and used as whole cell extracts (epitope mapping) or purified over a Dextrin Sepharose column (GE Healthcare) in one-step purification in 0.5 M sodium chloride, 20 mM Hepes-KOH pH 7.6, 1 mM EDTA and 5 mM dithiothreitol. Concentrated protein was dialysed against 200 mM sodium chloride, 20 mM Hepes-KOH, 1 mM EDTA and 5 mM dithiothreitol and stored in aliquots at −80°C.

The empty pGEX-2TK or pGEX-4T-2 vectors or those encoding a GST-testin variant were transformed in the E. coli BL21(DE3) pLYSstar strain. Bacteria were pre-cultured at 37°C for 4 h in Luria Bertani (LB) medium supplemented with 1% glucose. This pre-culture was diluted (1/25) in LB medium and grown at 37°C until an optical density of 0.6. Recombinant protein expression was induced by isopropyl-β-D-thiogalactopyranoside (1 mM) in LB medium that was supplemented with 10 μM zinc-acetate and grown at 16°C overnight. Bacterial cells were lysed in 25 mM Hepes (pH 7.6), 0.1 mM EDTA, 5 mM dithiothreitol, complete protease inhibitor cocktail (Roche) and 10.000 units/ml lysozyme (Amersham) and sonicated for 5 min on ice. GST or the GST-fusion protein was purified from the clarified lysate (from a 25 ml cell culture) by incubation for 2 h at 4°C with 25 μl glutathione sepharose^™ 4B (GE Healthcare) in buffer A (25 mM Hepes (pH 7.6), 150 mM NaCl, complete protease inhibitor cocktail). After extensive washing in buffer A, the resin was used in the binding assay. See also scheme Fig 2A.