DHRS6
PDB:2AG5
Revision
Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:DHRS6A-s001
Entry Clone Source:synthetic
SGC Clone Accession:
Tag:Histag with TEV cleavage site
Host:E.coli BL21 DE3
Construct
Prelude:
Sequence:mgsshhhhhhssgrenlyfqGHMGRLDGKVIILTAAAQGIGQAAALAFAREGAKVIATDINESKLQELEKYPGIQTRVLDVTKKKQIDQFANEVERLDVLFNVAGFVHHGTVLDCEEKDWDFSMNLNVRSMYLMIKAFLPKMLAQKSGNIINMSSVASSVKGVVNRCVYSTTKAAVIGLTKSVAADFIQQGIRCNCVCPGTVDTPSLQERIQARGNPEEARNDFLKRQKTGRFATAEEIAMLCVYLASDESAYVTGNPVIIDGGWSLGS
Vector:p11
Growth
Medium:
Antibiotics:
Procedure:The construct was expressed in E. coli in 1 L TB medium in the presence of 100 micro;g/mL of ampicillin at 37 o C. Cells were induced at 0.5 mM IPTG as soon as the OD reached 0.9, and temperature was shifted to 18 o C, and the culture was grown for 12 hrs. Cells were collected by centrifugation.
Purification
Procedure
Column 1 : Ni-NTA resin
Buffers (adjusted to pH 8.0): Lysis buffer: 5 mM Imidazole500mM NaCl, 50 mM HEPES, pH 7.5, 5% glycerol. Wash buffer: 30 mM Imidazole, 500 mM NaCl50mM, HEPES, pH 7.5, 5% glycerol. Elution Buffer: 250 mM Imidazole, 500 mM NaCl, 50 mM HEPES, pH 7.5, 5% glycerol.
Column 2: Superdex S200
Buffer: 10 mM HEPES, pH 7.5, 500 mM NaCl, 5% glycerol, 1mM TCEP
Procedure: Sample was loaded, washed with wash buffer and eluted in elution buffer. The protein was concentrated using an Amicon Ultra device.
Extraction
Procedure
Pellets were resuspended in 20 mL lysis buffer including Protease inhibitor (complete, Roche), lysed by French Press, and the solution was centrifuged to obtain a clear supernatant (30 min, 20.000 x g). Supernatants were processed in a 2 step chromatographic procedure.
Concentration:
Ligand
MassSpec:
Crystallization:Crystals were obtained using the following conditions: Vapour diffusion method, sitting drop, 293K, 30% PEG 5K, 0.2 M ammoniumsulfate, 0.1 M MES, pH 6.5.
NMR Spectroscopy:
Data Collection:Resolution: 1.86 Å , X-ray source: rotating anode (Rigaku FR-E SuperBright), single wavelength.
Data Processing:
