Gsh resin

Human SH3 domain of BIN1 with Glutathione S-transferase (GST) Tag (GST-SH3) was produced in E. coli BL21 using pGEX6P1 plasmid after induction with IPTG (1 mM) for 3 h at 37 °C. Then, cells were centrifuged at 7.500 g, lysate and GST-SH3 was purified using Glutathione Sepharose 4B beads (GSH-resin). The GST tag was cut by PreScission enzyme (GE Healthcare) on the beads and the flow through was recovered. Human M-DNM2 and Ub-DNM2 recombinant protein were produced in Sf9 insect cells-Novagen (Sigma–Aldrich Chimie S.a.r.l., Ref 71104-3) with the baculovirus system as follows^{57 (link)}. Baculovirus were produced after transfection with DNM2 pVL1392 plasmids. Sf9 cells were infected with viruses and grown for 3 days at 27 °C and centrifuged at 2000 g for 10 min. DNM2 recombinant protein was purified with GST-SH3 of BIN1 bound to GSH-resin (GE Healthcare) as the SH3 domain captures full length dynamin 2 through a high affinity interaction with its PRD (column preparation described below)^{58 (link)}. The analytical gel-filtration of DNM2 has been done using a column S200 10/300 and the buffer (20 mM Hepes, pH7.4; 5% Glycerol; 1.5 M NaCl; 1 mM EGTA; 1 mM DTT). Purity and quality of the proteins after elution was analyzed after separation with 12% SDS-PAGE gel follow by Coomassie staining.

The maltose binding protein (MBP)-fused cue domain of Rqt3 was expressed by using pETM40-Rqt3, and the GST-fused ubiquitin was expressed by using pGEX-Ub in E. coli Rosetta-gami 2 (DE3) cells. The each transformed cell was grown at 23 °C in LB medium until they reached an absorbance at 600 nm (A_600 
nm) of 0.6, isopropyl-b-D-thiogalactoside was added to a final concentration of 0.1 mM. The cells were grown for an additional 3 h and then collected by centrifugation and stored frozen at −80 °C. Frozen pellets were resuspended in buffer (25 mM Tris, pH 7.5, 100 mM NaCl, 1 mM MgCl₂, 0.01% NP-40 and 2 mM β-mercaptoethanol) with protease-inhibitor cocktail (Roche), and were broken by sonication on ice. The lysate was centrifuged at 39,000×g for 30 min at 4 °C. The supernatant fractions were used for the purification step. The MBP-cue domain and GST-ubiquitin were purified by amylose resin (NEB) or GSH resin (GE Healthcare), respectively, and the both columns were washed with PBS buffer, including 1% Triton X-100 before elution. MBP-cue domain was eluted by the addition of 50 mM maltose and then mixed with immobilized GST or GST-ubiquitin GSH resin, followed by incubation for overnight at 4 °C. The column was washed with PBS buffer including 1% Triton X-100 and eluted by 20 mM glutathione. The final elution was analyzed by SDS-PAGE.