Edta free protease inhibitors cocktail

Plasmids for human kinase c-Abl (pGEX-cAbl a.a. 83–531) and the YopH phosphatase and was obtained from the laboratory of John Kuriyan, as described (Seeliger et al, 2005 (link)). Both plasmids were transformed into BL21-DE3 cells in LB medium with streptomycin (50 μg/ml)/ampicillin (100 μg/ml). Cells were grown at 37°C until OD600 of 1.2 was reached and cooled for 1 h at 18°C with shaking. Expression was induced with 0.2 mM IPTG for 16 h (overnight) at 18°C. Cells were harvested by centrifugation at 7,000g at 4°C for 10 min and resuspended in 25 ml of cold TBS buffer (50 mM Tris–HCl, 500 mM NaCl, pH 8) with 5% glycerol and EDTA-free protease inhibitors cocktail (Roche). Cells were lysed by sonication and purified using Glutathione-Sepharose 4B (Cytiva) and eluted with 20 mM reduced glutathione in TBS. Eluted proteins were resolved by gel filtration on Superdex 200 16/60 (Cytiva) in TBS pH 7.5. GST-TcPINK1 (a.a. 121–570) was purified as described previously (Rasool et al, 2018 (link)), using a method identical to that described for rat Parkin above, with the exception that no ZnCl₂ was added in the medium.