Human small RNA 2-O-methyltransferase, methyltransferase domain, in complex with S-adenosyl-L-homocysteine

HENMT1

PDB:4XCX

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:GI:156564365
Entry Clone Source:MGC
SGC Clone Accession:
Tag:N-terminal: 6XHis-tag with integrated TEV protease site: MHHHHHHSSGRENLYFQ*G
Host:E.coli BL21 (DE3) codon plus RIL (Stratagen).

Construct

Prelude:
Sequence:gNFEEVPRETAIQFKPPLYRQRYQFVKNLVDQHEPKKVADLGCGDTSLLRLLKVNPCIELLVGVDINEDKLRWRGDSLAPFLGDFLKPRDLNLTITLYHGSVVERDSRLLGFDLITCIELIEHLDSGDLARFPEVVFGYLSPSMIVISTPNSEFNPLFPSVTLRDSDHKFEWTRMEFQTWALYVANRYDYSVEFTGVGEPPAGAENVGYCTQIGIFRKNGGKATESCLSEQHDQHVYKAVFTTSYPSLQQ
Vector:pET28-MHL

Growth

Medium:
Antibiotics:
Procedure:C1orf59 protein was expressed in E.coli BL21 (DE3) codon plus RIL in Terrific Broth (TB) in the presence of 50 μg/ml of kanamycin. Cells were grown at 37oC to an OD600 of 1.5 and 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 lysate was loaded onto 5 ml HiTrap column (GE Healthcare), charged with Ni2+. The column was washed with 10 CV of 20 mM Tris-HCl pH 8.0, containing 250 mM NaCl, 50 mM imidazole, 5% glycerol, and the protein was eluted with elution buffer (20 mM Tris-HCl pH 8.0, 250 mM NaCl, 250 mM imidazole, 5% glycerol). The protein was loaded on Superdex200 column (26x60) (GE Healthcare), equilibrated with 20 mM Tris-HCl buffer, pH 8.0, and 150 mM NaCl. The fractions containing C1orf59 were pooled and TEV protease was added to remove His-tag. The protein was further purified to homogeneity by ion-exchange chromatography on Source 30Q column (10x10) (GE Healthcare), equilibrated with buffer containing 20 mM Tris-HCl, pH 8.0, and eluted with linear gradient of NaCl up to 500 mM concentration (20 CV).

Enzymatic treatment: TEV

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 purification, the cell paste was thawed and resuspended in lysis buffer buffer (1X PBS, 250 mM NaCl, 2 mM β-mercaptoethanol, 5% glycerol, 0.1% CHAPS) with protease inhibitor (0.1 mM phenylmethyl sulfonyl fluoride, PMSF). The cells were lysed by passing through Microfluidizer (Microfluidics Corp.) at 20,000 psi.
Concentration:13.4 mg/mL
Ligand
MassSpec:The expected mass for C1orf59 is 28444.2 Da, measured mass is 284477.04 Da.
Crystallization:Purified C1orf59 protein (5 mg/mL) was complexed with S-adenosyl-L-homocysteine (SAH) (Sigma) at 1:5 molar ratio of protein:SAH and crystallized using hanging drop vapor diffusion method at 20 °C by mixing 1 μl of the protein solution with 1 μl of the reservoir solution containing 2.4 M NaCl, 0.1 M Tris-HCl, pH 8.6, 4% 1,3-propanediol.
NMR Spectroscopy:
Data Collection:
Data Processing: