PRDM2
PDB:2QPW
Revision
Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi:55953107
Entry Clone Source:MGC
SGC Clone Accession:
Tag:N-terminal: His-tag with integrated thrombin protease site:
MGSSHHHHHHSSGLVPRGS
Host:E.coli BL21 (DE3) codon plus RIL (Stratagene).
Construct
Prelude:
Sequence:gsNQNTTEPVAATETLAEVPEHVLRGLPEEVRLFPSAVDKTRIGVWATKPILKGKKFGPFVGDKKKRSQVKNNVYMWEVYYPNLGWMCIDATDPEKGNWLRYVNWACSGEEQNLFPLEINRAIYYKTLKPIAPGEELLVWYNGEDNPEI
Vector:pET28a-LIC
Growth
Medium:
Antibiotics:
Procedure:PRDM2 was expressed in E.coli BL21 (DE3) codon plus in M9 minimal medium in the presence of 50 μg/ml of kanamycin. Cell were grown at 37 degC to an OD600 of 0.8 and induced by isopropyl-1-thio-D-galactopyranoside (IPTG), final concentration 1 mM, in the presence of 50 mg/L of SeMet and incubated overnight at 15 degC.
Purification
Procedure
Column 1: DE52
Column 2: 5 ml HiTrap column (Amersham Biosciences)
Column 3: Superdex200 column (26x60) (Amersham Biosciences)
Column 4: Source 30Q column (10x10) (Amersham Biosciences)
The crude extract was cleared by centrifugation and passing through 20-ml DE52 column equilibrated in 20 mM Tris-HCl, pH 8.0, containing 250 mM NaCl and 5% glycerol. The lysate was loaded onto 5 ml HiTrap column (Amersham Biosciences), charged with Ni2+. The column was washed with 10 CV of wash buffer, and the protein was eluted with elution buffer . The protein was loaded on Superdex200 column (26x60) (Amersham Biosciences), equilibrated with 20 mM Tris-HCl buffer, pH 8.0, and 150 mM NaCl, at flow rate 4 ml/min. Thrombin (Sigma) was added to combined fractions containing PRDM2 and incubated overnight at 4 degC. The protein was further purified to homogeneity by ion-exchange chromatography on Source 30Q column (10x10) (Amersham Biosciences), equilibrated with buffer 20 mM Tris-HCl, pH 8.0, and eluted with linear gradient of NaCl up to 500 mM concentration (20CV). Purification yield was 16 mg of the protein per 1L of culture.
Extraction
Procedure
Cells were harvested by centrifugation at 7,000 rpm. The cell pellets were frozen in liquid nitrogen and stored at -80 degC. For the purification the cell paste was thawed and resuspended in lysis buffer with protease inhibitor (0.1mM phenylmethyl sulfonyl fluoride, PMSF). The cells were lysed by passing through Microfluidizer (Microfluidics Corp.) at 20,000 psi.
Concentration:21 mg/ml
Ligand
MassSpec:Expected MW for SeMet labelled protein is 18910.43 Da, measured mass is 18873.5451 Da.
Crystallization:Purified PRDM2 was crystallized using hanging drop vapor diffusion method at 20 °C by mixing 1.5 µl of the protein solution with 1.5 µl of the reservoir solution containing 22% PEG 5,000, 0.2 M ammonium sulfate, 0.1 M MES pH 7.0.
NMR Spectroscopy:
Data Collection:
Data Processing:
