RAB3B
PDB:3DZ8
Revision
Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:BC005035
Entry Clone Source:MGC AU35-H11
SGC Clone Accession:HPC085-A01
Tag:mhhhhhhssgrenlyfq*g
Host:BL21-CodonPlus(DE3)-V2R
Construct
Prelude:
Sequence:mhhhhhhssgrenlyfqgNFDYMFKLLIIGNSSVGKTSFLFRYADDTFTPAFVSTVGIDFKVKTVYRHEKRVKLQIWDTAGQERYRTITTAYYRGAMGFILMYDITNEESFNAVQDWATQIKTYSWDNAQVILVGNKCDMEEERVVPTEKGQLLAEQLGFDFFEASAKENISVRQAFERLVDAICDKMSDS
Vector:pET28-mhl (GI:134105571)
Growth
Medium:Terrific Broth
Antibiotics:
Procedure:LEX Bubbling. The target protein was expressed in E. coli by inoculating 100 mL of overnight culture grown in Luria-Bertani medium into a 1.8 L of Terrific Broth medium in the presence of 50 µg/mL kanamycin and 25 µg/mL chloramphenicol at 37 °reeC. When OD600 reached ~3.0, the temperature of the medium was lowered to 15 µC and the culture was induced with 0.5 mM IPTG. The cells were allowed to grow overnight before they were harvested and flash frozen in liquid nitrogen and stored at -80 °C.
Purification
Procedure
The lysate was centrifuged at 16,000 rpm for 60 minutes and the supernatant was mixed with 3 mL 50% Ni-NTA beads, and incubated at 4 °reeC for 2 hours on a roller drum. The supernatant was then passed through a gravity column (Poly-Prep, Bio-Rad, Catalog #731-1550) and the beads were washed using 50 mL washing buffer twice. The protein bound to beads were eluted using 15 mL elution buffer. The flow-through was collected and loaded onto Supderdex-75 26/60 gel filtration column. Eluted fractions were pooled and concentrated using amicon centrifugal filter (m.w. cut-off 10,000 ). The purity of the proteins was higher than 95% judged by SDS-PAGE.
Extraction
Procedure
Frozen cells from 3.6L TB culture were thawed and resuspended in 400 mL extraction buffer with freshly added 0.5% CHAPS and 10U/mL Benzonase (Sigma Catalog # E1014, 250U/µL), and supplemented with 1mL protease inhibitor cocktail (SIGMA Catalog # P8849), and lysed using sonication at 10 seconds 50% duty cycle for 8 minutes at 120W.
Concentration:18.0 mg/mL
Ligand
GDPMassSpec:Native: 22155.29, expected 22154.94
Crystallization:Crystallization was setup using sitting drops with Red Wings and SGC-I screens initially. 2D-plate xtals were seen at condition SG04, SD11 several days after setup.
Crystal used for data collection was grown at 2.0M (NH4)2SO4, 0.2M NaAc, 0.1M HEPES pH 7.5, 5% MPD.
Last updated by ytong 20080730
NMR Spectroscopy:
Data Collection:
Data Processing:
