DOT1L
PDB:3UWP
Revision
Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:GenBank: AF509504.1
Entry Clone Source:DNA 04-G3 (GeneScript)
SGC Clone Accession:DOT1L:JMC01M-E09:C206422
Tag:N-terminal His6-tag, removed by TEV
Host:BL21-V2R-pRARE
Construct
Prelude:sythetic gene
Sequence:MGEKLELRLKSPVGAEPAVYPWPLPVYDKHHDAAHEIIETIRWVCEEIPDLKLAMENYVLIDYDTKSFESMQRLCDKYNRAIDSIHQLWKGTTQPMKLNTRPSTGLLRHILQQVYNHSVTDPEKLNNYEPFSPEVYGETSFDLVAQMIDEIKMTDDDLFVDLGSGVGQVVLQVAAATNCKHHYGVEKADIPAKYAETMDREFRKWMKWYGKKHAEYTLERGDFLSEEWRERIANTSVIFVNNFAFGPEVDHQLKERFANMKEGGRIVSSKPFAPLNFRINSRNLSDIGTIMRVVELSPLKGSVSWTGKPVSYYLHTIDRTILENYFSSLKNPKLREEQEAARRRQQRESKSNAATPTKGPEGKVAGPADAPMDSGAEEEKAGAATVKKPSPSKARKKKLNKKGRKMAGRKRGRPKKMNTA
Vector:pET28-MHL
Growth
Medium:
Antibiotics:
Procedure:LEX Bubbling. The target protein was expressed in E. coli by inoculating 30 mL of overnight culture grown in Luria-Bertani medium into a 2 L of Terrific Broth medium in the presence of 50 ug/mL kanamycin and chloramphenicol at 37 degree. When OD600 reached ~2.0, the temperature of the medium was lowered to 15 degree and the culutre was induced with 0.5 mM IPTG. The cells were allowed to grow overnight before harvested by centrifugation (7,000 rpm 15min) and flash frozen in liquid nitrogen and stored at -80 degree.
Purification
Procedure
DOT1L (1-420) was purified by Ni-NTA column (Qiagen) and processed by in-house produced TEV protease to remove the His tag. The protein was then incubated in 50 mM Tris-HCl pH 8.0, 1 mM MgCl2 with benzonase nuclease for 2 hours at room temperature to remove DNA which binds to the C-terminal region of DOT1L (1-420). The filtered protein sample was diluted with 50 mM K2HPO4/ KH2PO4 pH 7.0, and further purified by HiTrap-SP (GE Healthcare). The protein was finally purified by gel filtration (Superdex 200, GE Healthcare)
Extraction
Procedure
2L cell pellet was resuspended in a total volume of 200 ml lysis buffer and the cells disrupted by sonication using Microfluidizer (Microfluidics M110-EH).
Concentration:16.0 mg/mL
Ligand
5-iodotubercidinMassSpec:The cut version native protein expected 47902.6, measured 47902.6
Crystallization:Purified protein DOT1L (1-420) was concentrated to 16 mg/mL in a buffer containing 20 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, and 1 mM TCEP. To obtain crystals of DOT1L/5-iodotubercidin, DOT1L (1-420) was mixed with 5-iodotubercidin by directly adding a 5 fold molar excess of compound to the protein solution, and the sitting drop vapor diffusion method was used at 18°C in a buffer containing 3.5 M sodium formate, and 100 mM sodium acetate, pH 4.6.
NMR Spectroscopy:
Data Collection:
Data Processing:
