DNM3
PDB:3L43
Revision
Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:SGC:05-C7
NM_015569.2
Entry Clone Source:Origene FB1503_F12
SGC Clone Accession:HPC09I-C03
Tag:mhhhhhhssgrenlyfq*g
Host:BL21-V2R-pRARE2
Construct
Prelude:DYN3:M6-S306
Tag was not removed
Sequence:mhhhhhhssgrenlyfqgMEELIPLVNRLQDAFSALGQSCLLELPQIAVVGGQSAGKSSVLENFVGRDFLPRGSGIVTRRPLVLQLITSKAEYAEFLHCKGKKFTDFDEVRLEIEAETDRVTGMNKGISSIPINLRVYSPHVLNLTLIDLPGITKVPVGDQPPDIEYQIREMIMQFITRENCLILAVTPANTDLANSDALKLAKEVDPQGLRTIGVITKLDLMDEGTDARDVLENKLLPLRRGYVGVVNRSQKDIDGKKDIKAAMLAERKFFLSHPAYRHIADRMGTPHLQKVLNQQLTNHIRDTLPNFRNKLQGQLLS
Vector:pET28-mhl (GI:134105571)
Growth
Medium:Antibiotics:Procedure:LEX Bubbling. The target protein was expressed in E. coli by inoculating 100 mL of overnight culture grown in Luria-Bertani medium into a 2 L of Terrific Broth medium in the presence of 50 µg/mL kanamycin and 25 µg/mL chloramphenicol at 37 °reeC. When OD600 reached ~3.0, the temperature of the medium was lowered to 15 °reeC and the culutre was induced with 0.5 mM IPTG. The cells were allowed to grow overnight before harvested and flash frozen in liquid nitrogen and stored at -80 °reeC.
Purification
ProcedureThe lysate was centrigued at 16,000 rpm for 1 hour and the supernatants were mixed with 8 mL 50% flurry of Ni-NTA beads and incubated at 4°reeC on rotary shaker for one hour. The mixture was then centrifuged at 3000 rpm for 3 min and the supernant discarded. The beads were then washed with washing buffer containing 20 mM Imidazole, and eluted with the elution buffer. The flow-trough was collected and further purifed by a Superdex-75 gel filtraton column pre-equilibrated with gel filtration buffer. Fractions containing the protein were collected and concentrated with Amicon Ultra-15 centrifugal filter (mw cut-off 10k). The purity of the preparation is tested by SDS-PAGE to be greater than 90%.
During purification, the tag was not removed.
Extraction
ProcedureFrozen cells from 6L TB culture were thawed and resuspended in 1000 mL extraction buffer with freshly added 0.5% CHAPS, and supplemented with 1.6 mL protease inhibitor cocktail (SIGMA Catalog # P8849), and 10 µL benzonase (Sigma Catalog # E1014, 250U/µL), 1mM PMSF/Benzamidine, and lysed using sonication at 120W for 6\'.
Concentration:25.9 mg/mL
LigandMassSpec:Native expected 35792.28, measured 35815.59 (delta +23.3, sample overloaded on mass)
Crystallization:Crystallization was setup using in situ proteolysis method in sitting drops with Red Wings and SGC-I screens initially. Crystals were found in RW-A3#2 and A10#2 (subtilisin), SGC-A9#1 (no protease) Diffracting crystals were found from initial screen drops.Crystal used for structure determination was grown in 25% PEG3350, 0.2M NaCl, 0.1 M HEPES, pH 7.5, with 1:100 Subtilisin A (w/w) in sitting drop setup (0.5uL+0.5uL).
Crystals grow to a mountable size with a few days.
0.9V well solution plus 0.1V 80% glycerol was used as cryoprotectant.
NMR Spectroscopy:Data Collection:Data Processing:
