DDX18
PDB:3LY5
Revision
Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi|38327634
Entry Clone Source:BC024739
SGC Clone Accession:DDX18A-s002
Tag:N-terminal hexahistidine tag with integrated TEV protease cleavage site: mhhhhhhssgvdlgtenlyfq*sm
Host:E.coli BL21(DE3) gold pRARE2, supplies tRNAs for rare codons.
Construct
Prelude:Sequence:mhhhhhhssgvdlgtenlyfq*smNNVEKPDNDEDESEVPSLPLGLTGAFEDTSFASLCNLVNENTLKAIKEMGFTNMTEIQHKSIRPLLEGRDLLAAAKTGSGKTLAFLIPAVELIVKLRFMPRNGTGVLILSPTRELAMQTFGVLKELMTHHVHTYGLIMGGSNRSAEAQKLGNGINIIVATPGRLLDHMQNTPGFMYKNLQCLVIDEADRILDVGFEEELKQIIKLLPTRRQTMLFSATQTRKVEDLARISLKKEPLYVG
Vector:pNIC-Bsa4
Growth
Medium:Antibiotics:Procedure:Cells from a glycerol stock were grown in 50 mL TB supplemented with 8 g/l glycerol, 100 µg/mL kanamycin and 34 µg/mL chloramphenicol at 37 °C overnight. The overnight culture (50 mL) was used to inoculate 3 l TB (divided into 4 x 0.75 l bottles) supplemented with 8 g/l glycerol, 50 µg/mL kanamycin and approximately 200 µl 204 Antifoam A6426 (Sigma) per bottle. The culture was grown in a LEX bioreactor system (Harbinger Biotechnology) at 37 °C until OD
600 nm had reached 1-2. The culture was down-tempered to 18 °C over a period of 1 hour before target expression was induced by addition of 0.5 mM IPTG. Expression was allowed to continue overnight and cells were harvested the following morning by centrifugation (4,400 x
g, 10 min, 4 °C). The resulting cell pellet (65 g wet cell weight) was then stored at -80 °C.
Purification
ProcedureColumns
IMAC: Ni-charged 5 mL HisTrap HP (GE Healthcare)Gel filtration column: HiLoad 16/60 Superdex 75 Prep Grade (GE Healthcare)
Procedure
The whole purification procedure was performed at 4 ºC. The protein sample was loaded onto an IMAC column previously equilibrated in Lysis buffer. The IMAC column was then washed with, first, 100 mL of IMAC wash1 buffer followed by 100 mL of IMAC wash2 buffer. Bound protein was eluted from the IMAC columns with IMAC elution buffer then loaded onto the gel filtration column equilibrated in GF buffer. Fractions containing the target protein were pooled.
Extraction
Procedure1.5 ml Extraction buffer per gram cell pellet and the 1 ml per 1.5 L culture Complete stock solution
1 was added and the cell pellets were resuspended at 4°C. The resuspended cells were stored in falcon tubes in the -80 freezer. Cells were disrupted by sonication (Vibra-Cell, Sonics) at 80% amplitude for 3 min effective time (pulsed 4s on, 4s off) and cell debris was removed by centrifugation (49,000 x
g, 40 min, 4 ºC). The supernatant was decanted and filtered through a 0.45 µm flask filter.
1Complete stock solution: 1 tablet Complete EDTA-free (protease inhibitor) and 8 µL Benzonase (2000 U) dissolved in 1 mL buffer
Concentration:LigandMassSpec:Crystallization:The Protein solution was incubated with 10 mM ADP and 10 mM MgCl2 for 1 hour. Crystals were obtained by the sitting drop vapour diffusion method in a 96-well plate. 0.1 µl protein solution (16.9 mg/ml) was mixed with 0.1 µl of well solution consisting of 0.1 M HEPES pH 6.6, 0.2 M sodium thiocyanate, 20% w/v PEG 3350. The plate was incubated at 20 ºC and crystals appeared after 14 days. The crystals were quickly transferred to a cryo solution consisting of 1 M HEPES pH 6.6, 0.2 M sodium thiocyanate, 22% w/v PEG 3350, 20% glycerol and flash frozen in liquid nitrogen.
NMR Spectroscopy:Data Collection:Diffraction data to 2.7 Å resolution was collected at Diamond beamline I04.
Data Processing:Intensity integration was performed by MOSFLM. Reflections were scaled with SCALA. The space group was P31 with cell dimensions a = 41.34 Å, b = 41.34 Å c = 230.54 Å, alpha = 90°, beta = 90°, gamma = 120°. The structure was solved by molecular replacement using MOLREP with the structure of human DEAD-BOX RNA helicase DDX10 as template (PDB: 2PL3). Structure refinement was performed with REFMAC 5.5.0102. R-factor = 0.246, R-free = 0.274.
