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FABP1: Human liver fatty acid binding protein 1

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PDB Code 2F73 Target Class Lipid signalling

Target FABP1A
Alias FABP1, FABPL, L-FABP
Disease Area/Function metabolism
Date Deposited 2005-11-30
Authors P.KURSULA, A.G.THORSELL, C.ARROWSMITH, H.BERGLUND, A.EDWARDS, M.EHN, S.FLODIN, S.GRASLUND, M.HAMMARSTROM, L.HOLMBERGSCHIAVONE, T.KOTENYOVA, P.NILSSON-EHLE, P.NORDLUND, T.NYMAN, D.OGG, C.PERSSON, J.SAGEMARK, P.STENMARK, M.SUNDSTROM, S.VAN DENBERG, J.WEIGELT, B.M.HALLBERG, STRUCTURAL GENOMICS CONSORTIUM(SGC)

Struc Details Tabs

Structure Details
Given the low aqueous solubility of fatty acids, their transport in the cell needs to be mediated by specific binding proteins. Liver fatty-acid-binding protein (FABP1) is found in high abundance in the hepatocyte cytosol, but associates also in the hepatocyte nucleus in a specific ligand-dependent manner. It facilitates the cellular uptake, transport and metabolism of fatty acids and is involved in the regulation of gene expressions and cell differentiation. FABP1 belongs to the family of intracellular lipid binding proteins, having a 10-stranded â-clam structure confining a lipid-binding cavity gated by two short anti-parallell helices; however, FABP1 is unique in this family in that the ligand pocket is unusually large and therefore capable of binding two molar equivalents of long-chain fatty acids and also larger ligands such as heme.

FABP1 binding and transport of peroxisome proliferators, especially leukotriene D4 antagonists, are implicated in side-effects of anti-inflammatory asthma therapy.

References

Cannon, JR & Eacho, PI (1999). Interaction of LY171883 and peroxisome proliferators with fatty–acid-binding protein isolated from rat liver (1991). Biochem. J., 280, 387-391.

Thompson, J, Reese-Wagoner, A & Banaszak, L.. Liver fatty acid binding protein: species variation and the accommodation of different ligands. BBA, 1441, 117-130.

Thompson J, Winter N, Terwey D, Bratt J & Banaszak L. (1997). The crystal structure of liver fatty-acid-binding protein. JBC, 272, 7140-7150.

Lawrence JW, Kroll, DJ & Eacho PI (2000). Ligand-dependent interaction of hepatic fatty-acid binding protein with the nucleus. J. Lipid. Res., 41, 1390-1401.

Materials & Methods
StructureFABP1
PDB Code2F73
Entry clone accession BC032801
Entry clone source MGC
Tag N-terminal hexahistidine tag with integrated TEV protease cleavage site: mhhhhhhssgvdlgtenlyfq*s(m).
Construct sequencemhhhhhhssgvdlgtenlyfqsMSFSGKYQLQSQENFEAFMKAIGLPEELIQKGKDIKGVSEIVQNGKHFKFTITAGSKV
IQNEFTVGEECELETMTGEKVKTVVQLEGDNKLVTTFKNIKSVTELNGDIITNTMTLGDIVFKRISKRI
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.

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