ARF5
PDB:2B6H
Revision
Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NP_001653
Entry Clone Source:MGC
SGC Clone Accession:
Tag:N-terminal: His-tag with integrated thrombin protease site before the last Ser: MGSSHHHHHHSSGLVPRGS
Host:E. coli BL21 (DE3)
Construct
Prelude:
Sequence:mgsshhhhhhssglvprgsLFSRIFGKKQMRILMVGLDAAGKTTILYKLKLGEIVTTIPTIGFNVETVEYKNICFTVWDVGGQDKIRPLWRHYFQNTQGLIFVVDSNDRERVQESADELQKMLQEDELRDAVLLVFANKQDMPNAMPVSELTDKLGLQHLRSRTWYVQATCATQGTGLYDGLDWLSHELSKR
Vector:p28a-LIC
Growth
Medium:
Antibiotics:
Procedure:We prepared the seeds by inoculating freshly transforming E. coli cells (BL21 DE3) into 80 mL of Luria-Bertani medium. After overnight growth, all of the seeds were inoculated into 1.8 L of Terrific Broth medium in the presence of 50 µg/mL of kanamycin at 37ºC and grown to an OD600 of 2.28. Cells were then induced by isopropyl-1-thio-D-galactopyranoside at the final concentration of 1.5 mM and grown overnight at 20ºC in the SGC LEX bubbling system.
Purification
Procedure
The supernatant was passed through DE52 (Whatman) column equilibrated with the binding buffer and then loaded onto 3 mL Ni-NTA column (Qiagen) equilibrated with the same binding buffer at 4ºC. The Ni-NTA column was washed with 150 mL of the wash buffer (10mM Tris pH7.5, 0.5 M NaCl, 5% glycerol, 30 mM imidazole) and the protein was eluted with 15 mL of the elution buffer (10mM Tris pH7.5, 0.5 M NaCl, 5% glycerol, 250 mM imidazole). The eluate was dialyzed overnight against a buffer containing 10 mM Tris pH 7.5, 0.5 M NaCl, 5% glycerol. The protein concentration was estimated based on Bradford assay. Five molar equivalents of GDP, 10 mM DTT and 5 mM MgCl2 were added to the purified protein before concentration. The protein was concentrated using an Amicon Ultra centrifugal filter to the final volume of 1 mL and the concentration of 20.0 mg/mL. About 20 mg of protein was obtained from 1.8 L of cell culture.
Extraction
Procedure
Cultures were centrifuged and the cell pellets were suspended in 100 mL of the binding buffer (10 mM Tris pH 7.5, 0.5 M NaCl, 5% glycerol, 5 mM imidazole) with a protease inhibitor cocktail (0.1 mM M benzamidine-HCl and 0.1 mM phenylmethyl sulfonyl fluoride) and flash frozen. The thawed cell pellet was lysed by a combination of 0.5% CHAPS (Sigma) and sonication. The lysate was centrifuged at 15000 rpm for 30 min and the supernatant was used for subsequent steps of purification.
Concentration:
Ligand
MassSpec:
Crystallization:Purified ARF5 was crystallized using the sitting drop vapor diffusion method at room temperature. Crystals grew in one day when the protein (20 mg/mL) was mixed with the reservoir solution in a 1:1 volume ratio, and the drop was equilibrated against a reservoir solution containing 25% PEG 4K, 0.2 M AmSO4, 0.1 M Tris pH 8.5, 3% w/v 6-aminocaproic acid at 4oC. The crystals were flash frozen with the mother liquor with 15% glycerol.
NMR Spectroscopy:
Data Collection:
Data Processing:
