ZFYVE9C
PDB:4BKW
Revision
Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:GI: BC032680
Entry Clone Source:MGC
SGC Clone Accession:ZFYVE9C-c009
Tag:MGHHHHHHSSGVDLGTENLYFQ. N-terminal hexahistidine tag cleavable by TEV protease.
Host:BL21 (DE3)-R3-pRARE2. Phage-resistant derivative of BL21 (DE3), with pRARE2 plasmid encoding rare codon tRNAs (chloramphenicol-resistant).
Construct
Prelude:
Sequence:MHHHHHHSSGVDLGTENLYFQSMNLIPEDGLPPILISTGVKGDYAVEEKPSQISVMQQLEDGGPDPLVFVLNANLLSMVKIVNYVNRKCWCFTTKGMHAVGQSEIVILLQCLPDEKCLPKDIFNHFVQLYRDALAGNVVSNLGHSFFSQSFLGSKEHGGFLYVTSTYQSLQDLVLPTPPYLFGILIQKWETPWAKVFPIRLMLRLGAEYRLYPCPLFSVRFRKPLFGETGHTIMNLLADFRNYQYTLPVVQGLVVDMEVRKTSIKIPSNRYNEMMKAMNKSNEHVLAGGACFNEKADSHLVCVQNDDGNYQTQAISIHNQPRKVTGASFFVFSGALKSSSGYLAKSSIVEDGVMVQITAENMDSLRQALREMKDFTITCGKADAEEPQEHIHIQWVDDDKNVSKGVVSPIDGKSMETITNVKIFHGSEYKANGKVIRWTEVFFLENDDQHNCLSDPADHSRLTEHVAKAFCLALCPHLKLLKEDGMTKLGLRVTLDSDQVGYQAGSNGQPLPSQYMNDLDSALVPVIHGGACQLSEGPVVMELIFYILENIV
Vector:pNIC28-Bsa4. T7/lac regulated, N-terminal His-tag, TEV, LIC cloning using BsaI cleavage/T4 polymerase, SacB stuffer fragment, pET28 backbone.
Growth
Medium:A glycerol stock was used to inoculate a 100ml starter culture containing LB media with 50µg/ml Kanamycin and 34µg/ml Chloramphenicol. The starter culture was grown overnight at 37°C with shaking at 200 rpm. The following morning, two flasks containing 1L LB/Kanamycin were each inoculated with 10 ml of the starter culture. Cultures were incubated at 37°C with shaking at 170 rpm until an OD600nm ≥ 0.4 was reached. The flasks were then cooled down to 18°C to an OD600nm ≥ 0.7 and added 0.4mM IPTG to induce protein expression overnight. Cells were harvested by centrifugation at 5000 rpm at 4°C for 15 min. Cell pellets from each flask were resuspended in 25ml Binding buffer (50mM HEPES, pH 7.5; 500mM NaCl; 5% Glycerol; 5mM Imidazole).
Antibiotics:
Procedure:
Purification
Buffers
Procedure
Extraction
Buffers
Procedure
Extraction buffer, extraction method: The frozen cells were thawed. The cells were lysed by ultrasonication over 15 min with the sonicator pulsing ON for 5 sec and OFF for 10. A final concentration of 0.15% PEI was added to the lysate. The cell lysate was spun down by centrifugation at 21K rpm at 4°C for 1 h. The supernatant was recovered for purification. Column 1: Ni-Affinity Chromatography. 4 ml of 50 % Ni-sepharose slurry was applied onto a 1.5 x 10 cm column. The column was equilibrated with binding buffer (25ml). Buffers: Binding buffer: 50 mM Hepes, pH 7.5; 500 mM NaCl; 5% Glycerol; 5 mM imidazole 0.5mM TCEP Wash buffer: 50 mM Hepes, pH 7.5; 500 mM NaCl; 5% Glycerol; 25 mM imidazole 0.5mM TCEPElution buffer: 50 mM HEPES, pH 7.5; 500 mM NaCl; 5% Glycerol; 50 and 250 mM imidazole 0.5mM TCEPProcedure: The supernatant following centrifugation was applied by gravity flow onto the Ni-sepharose column. The bound protein was then washed with 50ml binding buffer and subsequently with 30 ml wash buffer. ZFYVE9C protein was then eluted by applying a step gradient of imidazole  using 5 ml portions of elution buffer with increasing concentration of imidazole (1 x 50 mM, 3 x 250 mM). Elution fractions were analyzed by SDS PAGE and the 3 x 250 mM imidazole fractions were kept and pooled. 10 mM DTT was added for overnight storage at 4C.Enzymatic treatment: TEV protease cleavage. Fractions containing ZFYVE9C were treated with TEV protease overnight at 4°C.Column 2: Size Exclusion Chromatography  S200 HiLoad 16/60 Superdex run on ÄKTA-ExpressGel Filtration buffer: 300 mM NaCl, 50 mM HEPES pH 7.5, 0.5 mM TCEP, pH 7.5Procedure: The Superdex S200 column was first equilibrated with Gel Filtration buffer. The protein fraction from above step was concentrated to Concentration:
Ligand
MassSpec:
Crystallization:Protein was concentrated down to 500ul and diluted to 10ml in gel filtration buffer before being reconcentrated down to 12.6mg/ml. Crystals were grown at 20°C in 300nl sitting drops mixing 200nl protein solution with 100nl reservoir solution containing 30% PEG 3350; 0.1M bis-tris pH6.5; 0.15M magnesium chloride. On mounting crystals were cryo-protected with an additional 25% ethylene glycol.
NMR Spectroscopy:
Data Collection:2.53 Ã
resolution; X-ray source: Diamond Light Source, station I03, using monochromatic radiation at wavelength 0.9795 Ã
Data Processing:
