Purification and Characterization of Human Dynamin-2 and BIN1 SH3 Domain

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.