Target: mNNMT
PDB:2I62
Revision
Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:GI:6754866
Entry Clone Source:MGC
SGC Clone Accession:
Tag:N-terminal: His-tag with integrated TEV protease site: MHHHHHHSSGRENLYFQ*G
Host:E.coli BL21 (DE3) codon plus RIL (Stratagen).
Construct
Prelude:
Sequence:gMESGFTSKDTYLSHFNPRDYLEKYYSFGSRHCAENEILRHLLKNLFKIFCLGAVKGELLIDIGSGPTIYQLLSACESFTEIIVSDYTDQNLWELQKWLKKEPGAFDWSPVVTYVCDLEGNRMKGPEKEEKLRRAIKQVLKCDVTQSQPLGGVSLPPADCLLSTLCLDAACPDLPAYRTALRNLGSLLKPGGFLVMVDALKSSYYMIGEQKFSSLPLGWETVRDAVEEAGYTIEQFEVISQNYSSTTSNNEGLFSLVGRKPGRSE
Vector:p28a-MHL
Growth
Medium:
Antibiotics:
Procedure:Mouse NNMT was expressed in E.coli BL21 (DE3) codon plus RIL in Terrific Broth (TB) in the presence of 50 µg/ml of kanamycin at 37oC to an OD600 of 1.5. Cells were then induced by isopropyl-1-thio-D-galactopyranoside (IPTG), final concentration 1 mM, and incubated overnight at 15oC.
Purification
Procedure
The crude extract was cleared by centrifugation. The clarified lysate was loaded onto 5 ml HiTrap Chelating column (Amersham Biosciences), charged with Ni2+. The column was washed with 10 CV of 20 mM Tris-HCl buffer, pH 8.0, containing 250 mM NaCl, 5% glycerol, 50 mM imidazole, and the protein was eluted with elution buffer (20 mM Tris-HCl, pH 8.0, 250 mM NaCl, 5% glycerol, 250 mM imidazole). Eluted protein was dialyzed overnight against 20 mM Tris-HCl, pH 8.0, 250 mM NaCl, 5% glycerol, 5 mM ß-mercaptoethanol in the presence of TEV protease to remove His-tag. 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 and 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 2.2 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°C. For the purification the cell paste was thawed and resuspended in lysis buffer (1 XPBS, 0.25 M NaCl, 5 mM imidazol, 2 mM ß-mercaptoethanol, 5% glycerol) with protease inhibitor (0.1mM phenylmethyl sulfonyl fluoride, PMSF). The cells were lysed by passing through Microfluidizer (Microfluidics Corp.) at 20,000 psi.
Concentration:11.3 mg/ml
Ligand
MassSpec:expected MW is 29654.78 Da, measured MW is 29654.86 Da.
Crystallization:Purified mouse NNMT (9 mg/ml) was complexed with S-adenosyl-L-homocysteine (SAH) (Sigma) at 1:5 molar ratio of protein: SAH and crystallized using the sitting drop vapor diffusion method at 20 °C by mixing 1 µl of the protein solution with 1 µl of the reservoir solution containing 10% PEG 4000, 0.1 M Na OAc, 0.1 M NaOAc pH4.6.
NMR Spectroscopy:
Data Collection:
Data Processing: