Gstrap column

The plasmid pGEX-4T-1-3xFlag-ERK2 with a GST-tag and triple Flag-tag at the N-terminus was obtained from Addgene (plasmid no. 47573; gift from Kevin Janes). The 3xFlag tag was inserted at BamHI site, and a new BamHI site was regenerated at its downstream. erk2 gene was inserted between BamHI and SalI sites of this pGEX-4T1 vector. GST-Flag-ERK2 protein was expressed in E. coli Rosetta 2 (DE3) cells. The overnight cultures were inoculated in a 1L fresh LB media with 100 μg/ml ampicillin at 37 °C. Cells were induced with 0.5 mM IPTG until OD₆₀₀ reaches 0.7 and incubated at 18 °C for further18 h. Cell pellets were harvested and then lysed in a 25 ml ice-cold PBS buffer with 1X protease inhibitor. The supernatants were centrifugated at 76,000 x g for 30 mins at 4 °C, filtered by centrifugal filters and then applied onto a GSTrap column (GE healthcare) at a flow rate of 1.5 mL/min. The loaded column was washed with 10 column volumes of PBS to remove non-bound components and the purified GST-Flag-ERK2 protein was recovered from the column with a elution buffer (1xPBS buffer containing 10 mM reduced glutathione and 1X protease inhibitor). The fractions with desired protein were analysed by SDS-PAGE and verified by Xevo mass spectrometer.

BL21(DE3) transformed with pGEX-6P expressing GST-tagged mortalin-PBD, or PBD-V482F was grown in Luria-Bertani medium at 37°C for 16 hours. Protein expression was induced with 1 mM isopropyl-β-D-thiogalactopyranoside for 16 hours at 16°C. Cell pellets were collected by centrifugation, lysed in 50 mM Tris (pH 7.5)/150 mM NaCl/0.5% Triton X-100/1 mM EDTA/1 mM dithiothreitol/1 mM phenylmethylsulfonyl fluoride, and sonicated. Supernatants were loaded onto 5 mL GSTrap column (GE Healthcare), washed with 50 mM Tris (pH 7.5)/500 mM NaCl/0.5% Triton X-100/1 mM EDTA, and eluted by 0–100% gradients of 50 mM Tris (pH 7.5)/50 mM NaCl/0.5% Triton X-100/1 mM EDTA/10 mM DTT/50 mM reduced glutathione/10 % glycerol using AKTA FPLC (GE Healthcare). Elutes were dialyzed (8 kDa cutoff) in 20 mM Tris (pH 7.5)/100 mM NaCl/0.05% Triton X-100/5 mM β-mercaptoethanol at 4°C for 16 hours prior to PreScission protease (GE Healthcare) treatment. Proteolysis fractions were separated by glutathione-agarose column (Gold Biotechnology, St. Louis, MO) and the flow-through was concentrated by 30 kDa cutoff centricon (EMD Millipore). Purity of recombinant proteins was determined by SDS-PAGE using Stain-free fluorescent gel (Bio-Rad). Full length recombinant mortalin (65 (link)) was obtained from Abdussalam Azem (Tel Aviv Univ).