RAB4B-GDP

PDB:2O52

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NM_016154
Entry Clone Source:Codon Devices
SGC Clone Accession:
Tag:N-terminal hexahistidine tag
Host:E.coli. BL21 (DE3) codon(+) RIL

Construct

Prelude:
Sequence:

mgsshhhhhhssglvprgsIWSDFLFKFLVIGSAGTGKSCLLHQFIENKFKQDSNHTIGVEFGSRVVNVGGKTVKLQIWDTAGQERFRSVTRSYYRGAAGALLVYDITSRETYNSLAAWLTDARTLASPNIVVILCGNKKDLDPEREVTFLEASRFAQENELMFLETSALTGENVEEAFLKCARTILNKIDSGELDPERM

Vector:p28a-thrombin-lic

Growth

Medium:Terrific Broth
Antibiotics:
Procedure:The target protein was expressed in E.coli. BL21 (DE3) coden (+) RIL containing the plasmid. The 100 ml overnight culture in Luria-Bertani medium was inoculated into 1.8 L of Terrific Broth medium in the presence of 50 µg/mL of kanamycin and 50 µg/mL chloramphenicol at 37 ºC and grown to an OD600 ~ 3.0 in the SGC LEX bubbling system. The culture was induced by isopropyl-1-thio-D-galactopyranoside at the final concentration of 1.0 mM and grown overnight at 18 ºC. The culture was harvested with centrifugation. Pellets were flash frozen and stored at -80 ºC.

Purification

Procedure
Column 1: Ni-NTA beads
Procedure: 2.5 ml of Ni-NTA suspension solution was added into 100 ml cell lysis supernatant solution. The mixture was shaken for 1 hour at 4 ºC. Beads were collected by centrifugation at 2500 rpm for 5 minutes. Beads were washed with 75 ml washing buffer, then collected by centrifugation. Protein was eluted with 15 ml elution buffer.

Column 2 : Size exclusion chromatography (Superdex 75 26/60)
The fractions eluted off the Ni-affinity column were applied to a Superdex 75 column (26/60) equilibrated in SEC buffer at a flow rate of 2.0 ml/min. Eluted fractions were 95% pure as judged by SDS-PAGE.

Protein concentration: Amicon ultra centrifugal filter with a 5kDa cut off in SEC-buffer

Extraction

Procedure
Cells were thawed and resuspended in 100 mL binding buffer (10 mM Tris pH 7.5, 0.5 M NaCl, 5 mM imidazole) with 0.5% CHAPS (Sigma) and 1 mM phenylmethyl sulfonyl fluoride (PMSF), 0.5% (v/v) protease inhibitor cocktail (Sigma), 1 mM Benzamidine, 1600 units Benzonase (Sigma), and lysed with a microfluidizer. The lysate was centrifuged at 16000 rpm for 45 min and the supernatant was used for subsequent steps of purification. All the extraction steps were carried out at 4 ºC.
Concentration:
Ligand
MassSpec:
Crystallization:Crystals were obtained using the vapor diffusion method. The protein solution (17 mg/ml) contained a 5 fold molar of GDP and MgCl₂. 0.5 µl of the concentrated protein was mixed with 0.5 µl of a well solution containing 18% PEG8000, 0.2 M Mg(OAc)₂, 0.1 M NaCaco, pH 6.50. Crystals appeared after three days at 18 ºC.
NMR Spectroscopy:
Data Collection:Crystals were cryo-protected using a mixture of 50% mineral oil and 50% parotone, and flash frozen in liquid nitrogen. Diffraction data were collected at Rigaku FR-E with Rigaku R-AXIS IV++ detector to 2.2 Å.
Data Processing: