The glutathione S-transferase (GST)-Bcl-GL recombinant protein was expressed in Escherichia coli strain BL21 codon-plus RIL competent cells (Stratagene).
Purification of the recombinant proteins was performed using Glutathione Sepharose 4B beads (GE Healthcare) under nondenaturing conditions according to the supplier's instructions.
For confirmation of direct binding of BCL-GL and MELK, we removed GST from GST-fused BCL-GL protein using PreScission protease (GE Healthcare) according to the supplier's instructions.Recombinant proteins of human APPL1 N-terminal fragments including the BAR (residues 5−265) and BAR-PH domains (residues 5−385) were expressed in Escherichia coli, then purified using His tag affinity chromatography.
His-tagged proteins of APPL1 (5−265) and APPL1 (5−385) were expressed as soluble recombinant proteins in BL21 StarTM (DE3) strain of E.
coli (Invitrogen), and cells were harvested after induction with 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 8 h at 25°C.
The cells were lysed with lysozyme, and the lysate supernatant was purified with His-SelectTM affinity beads (Sigma).
In both cases, the His tag was removed with thrombin.
Recombinant proteins of human Rab5a variants (GenBank ID: NM_004162), human Rab21 (BC021901), and human Rab22a (NM_020673) fused with an N-terminal GST were expressed in BL21 E.
coli and purified with GST-affinity chromatography.
The sample was concentrated to ∼20 mg ml−1 and stored in 1 × phosphate-buffered saline (PBS) with 0.1% (v/v) βME at −80°C.
Recombinant protein of human rabaptin5 (551–862) (GenBank ID: CAA62580) was expressed and purified as described previously (Zhai et al, 2003); two additional point mutations, C719S and C734S, were introduced to reduce aggregation.Soluble fractions were filtered with 0.8 μm syringe filters and applied into a Ni-NTA affinity column pre-equilibrated with 30 mM Tris-HCl (pH 8.0), 50 mM NaCl, 5 mM β-mercaptoethanol.
Target protein complexes (the Aα subunit with GST-tag and SV40 ST with His-tag) were eluted with elution buffer (30 mM Tris-HCl [pH 8.0], 50 mM NaCl, 300 mM imidazole, 5 mM β-mercaptoethanol) and dialyzed overnight at 4 °C in 30 mM Tris-HCl (pH 8.0), 50 mM NaCl, 5 mM DTT.
Dialyzed protein was applied to a GST affinity column to remove free SV40 ST, and on-column cleavage with TEV protease was performed at 4 °C overnight.
The flow-through fraction of the GST column was reapplied into the Ni-NTA column to remove cleaved His-tag and TEV protease.