Useful Information

Please contact us for any questions or request for reagents.

Materials and Methods: FABP1 (2F73)

Click on the iSee icon to launch an enhanced annotation with interactive 3D representations and animated transitions. Please note that a web plugin (activeICM) is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available.

Structure	FABP1
PDB Code	2F73
Entry clone accession	BC032801
Entry clone source	MGC
Tag	N-terminal hexahistidine tag with integrated TEV protease cleavage site: mhhhhhhssgvdlgtenlyfq*s(m).
Construct sequence	mhhhhhhssgvdlgtenlyfqsMSFSGKYQLQSQENFEAFMKAIGLPEELIQKGKDIKGVSEIVQNGKHFKFTITAGSKVIQNEFTVGEECELETMTGEKVKTVVQLEGDNKLVTTFKNIKSVTELN GDIITNTMTLGDIVFKRISKRI
Vector	pNIC-Bsa4
Expression host	E. coli Bl21(DE3)
Growth method	30 µL competent Bl21(DE3) cells (Novagen) were transformed with 1 µL plasmid. Held 30min on ice, heat-shocked in 42 degree waterbath for 45sec at 42°C. Held on ice for 2 min. Added 100µL SOC and incubated in shaker for 1 h. Cells were plated on LA plates with 50 mg/l Kanamycin and 0.2% glucose. Glycerol stocks were made by adding 5 colonies to 1mL phosphate buffered TB with 50 mg/l Kanamycin. 150 ul culture was taken out at mid-logphase (3 hours growth at 37 degrees) and mixed with sterile glycerol to a final concentration of 20% and then frozen at -80. The glycerol stock was used to inoculate 4 mL of SOC + Kan 50 mg/l in a 50 mL Falcon tube. The inoculation culture was shaken at 37 degrees for 3 hours when it was added to a TunAir flask (Shelton Scientific) with 750 mL of phosphate buffered TB with 50 mg/l Kanamycin. The culture was incubated at 37°C, until OD₆₀₀ reached of approximately 1.4. The temperature was lowered to 18°C and the culture was induced with 0.5 mM IPTG for 15 hours.
Extraction procedure	Cells were harvested by centrifugation (OD₆₀₀ 13.4; WCW 18 g) and pellets were resuspended in 30 mL of lysis buffer (50mM HEPES pH 7.5, 500mM NaCl, 10% glycerol, 10 mM Imidazole, 0.5 mm TCEP and 1 tablet Complete EDTA-free protease inhibitor (Roche Biosciences)). A knife edge of lysozyme (Sigma) was added before freezing at -20. After thawing, 4 µL of a 250 U/µl benzonase (Novagen) stock solution was added and lysis buffer was added to a total volume of 70 mL. Cells were then disrupted by high pressure homogenization with a high-pressure homogenizer (Stansted) (4 passes) prior to centrifugation for 30 min at 49000 g in a Sorvall SA-800 rotor. The soluble fraction was decanted and filtered through 0.22 µm.
Purification procedure	Buffers: 50 mM HEPES, pH 7.5, 10 mM Imidazole, 500 mM NaCl, 10% glycerol, 0.5 mM TCEP (IMAC Bind/Wash1 Buffer); 50 mM HEPES, pH 7.5, 50 mM Imidazole, 500 mM NaCl, 10% glycerol, 0.5 mM TCEP (IMAC Wash2 Buffer); 50 mM HEPES, pH 7.5, 400 mM Imidazole, 500 mM NaCl, 10% glycerol, 0.5 mM TCEP (IMAC Elution Buffer). Columns: 1 mL Hi-Trap Chelating (Ni-charged). (GE Healthcare). Superdex 75 HiLoad 16/60 (GE Healthcare). Procedure: The sample was purified automatically on an ÄKTA-Xpress (GE Healthcare). Briefly, sample was loaded on the IMAC column, eluted in a storage loop and then loaded on the gel filtration column. Elution fractions were pooled based on SDS-PAGE analysis. Protein was estimated by SDS-PAGE analysis to be more than 95% pure. Fresh TCEP was added to the pooled samples so that the concentration of TCEP was 2 mM. Concentration was performed by use of Amicon Ultra 15 (Millipore) with 10 000 MW CO. Centrifugation was performed at 15 deg in swing-out buckets, in 5 minutes intervals and maximum 15 minutes, at 3000 g. Yield of purified protein per liter of culture was 4.9 mg.
Crystallization	Buffers: 50 mM HEPES, pH 7.5, 10 mM Imidazole, 500 mM NaCl, 10% glycerol, 0.5 mM TCEP (IMAC Bind/Wash1 Buffer); 50 mM HEPES, pH 7.5, 50 mM Imidazole, 500 mM NaCl, 10% glycerol, 0.5 mM TCEP (IMAC Wash2 Buffer); 50 mM HEPES, pH 7.5, 400 mM Imidazole, 500 mM NaCl, 10% glycerol, 0.5 mM TCEP (IMAC Elution Buffer). Columns: 1 mL Hi-Trap Chelating (Ni-charged). (GE Healthcare). Superdex 75 HiLoad 16/60 (GE Healthcare). Procedure: The sample was purified automatically on an ÄKTA-Xpress (GE Healthcare). Briefly, sample was loaded on the IMAC column, eluted in a storage loop and then loaded on the gel filtration column. Elution fractions were pooled based on SDS-PAGE analysis. Protein was estimated by SDS-PAGE analysis to be more than 95% pure. Fresh TCEP was added to the pooled samples so that the concentration of TCEP was 2 mM. Concentration was performed by use of Amicon Ultra 15 (Millipore) with 10 000 MW CO. Centrifugation was performed at 15 deg in swing-out buckets, in 5 minutes intervals and maximum 15 minutes, at 3000 g. Yield of purified protein per liter of culture was 4.9 mg.