AK2
PDB:2C9Y
Revision
Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NM_001625.2
Entry Clone Source:TKC, MGC
SGC Clone Accession:Tag:Tag sequence: mhhhhhhssgvdlgtenlyfq*s(m). TEV-cleavable (*) N-terminal his6 tag.
Host:BL21 (DE3)
Construct
Prelude:Sequence:mhhhhhhssgvdlgtenlyfqsmAPSVPAEPEYPKGIRAVLLGPPGAGKGTQAPRLAENFCVCHLATGDMLRAMVASGSELGKKLKATMDAGKLVSDEMVVELIEKNLETPLCKNGFLLDGFPRTVRQAEMLDDLMEKRKEKLDSVIEFSIPDSLLIRRITGRLIHPKSGRSYHEEFNPPKEPMKDDITGEPLIRRSDDNEKALKIRLQAYHTQTTPLIEYYRKRGIHSAIDASQTPDVVFASILAAFSKATCKDLVMFILQ
Vector:pLIC- SGC1
Growth
Medium:Antibiotics:Procedure:A starter cultures was grown from a freshly transformed colony in 10 mL LB, 0.1 mg/mL ampiciline. This culture was diluted 1:1000 in fresh media and was grown at 37°C to an OD 600 of 0.4 and subsequently transferred to 18°C. Expression was induced at an OD600 of 0.6 - 0.7 using 1 mM IPTG (final concentration). Cells were harvested after 4h by centrifugation (15min, 6000rpm on a JLA 8.100 rotor), transferred to 50-mL tubes, and frozen at -20°C.
Purification
ProcedureColumn 1: A DE52 column (10gr in 100mL of 2.5M NaCl) was equilibrated with 100mL of Loading Buffer. A 5mL NiNTA column was equilibrated with 20mL of Loading Buffer
Buffers: Loading buffer: 50 mM Hepes, pH 7.5, 500 mM NaCl, 5% glycerol. Wash buffer: 50 mM Hepes, pH 7.5, 500 mM NaCl, 20 mM imidazole, 5% glycerol. Elution buffer: 50 mM Hepes, pH 7.5, 500 mM NaCl, 50-250 mM imidazole (step elution), 5% glycerol.
Procedure: A DE52 column (10gr suspended in 100mL of 2.5M NaCl) was equilibrated with 100mL of Loading Buffer. A 5mL NiNTA column was equilibrated with 20mL of loading buffer. The lysed sample was applied to the DE-52 column and washed through with 50 mL loading buffer. The flow through was applied to the 5 mL Ni-NTA column which was washed with 2x10mL of wash buffer and eluted with elution buffer in 5 mL aliquots (Step elution using 50, 100, 150, 200 and 250 mM imidazole in the Elution Buffer)
Column 2: SEC
Buffers: Gel Filtration Buffer: 10mM HEPES, pH 7.5, 100mM NaCl
Procedure: AKTA-prime
Fractions containing AK2 collected from IMAC and treated with TEV protease overnight (identified by SDS PAGE ) were concentrated to about 1.5mL and directly applied to a S75 16/60 column equilibrated in 10 mM Hepes pH 7.5, 100 m NaCl. The flow rate was 1mL/min and the pure protein eluted at 60-70min.
Enzymatic treatment: Treated the IMAC elution(s) with TEV protease overnight at 4°C.
Extraction
ProcedureExtraction buffer: 50 mM HEPES, pH 7.5, 500 mM NaCl, 5% Glycerol. The cell pellets (5 gr wet wt) were re-suspended in 50 mL extraction buffer containing a Protease Inhibitor cocktail tablet (Roche), and lysed in a high pressure cell disrupter. The supernatant was centrifuged for 30 minutes at 35k g in a JA 25.5 rotor at 4°C.
Concentration:LigandMassSpec:Crystallization:Crystals were grown at 4°C in 600nl sitting drops mixing 200nl of AK2 (8.5 mg/mL in 10mM Hepes pH 7.5, 100mM NaCl ,10mM DTT containing 1 mM Ap4A) with 400nl of a solution containing 28% PEG 3350, 100 mM Bis Tris pH 5.7. Crystals were frozen by transfer into liquid nitrogen in crystallization buffer with additional 25% ethylene glycol.
NMR Spectroscopy:Data Collection:Data Processing:
