ABSTRACT
The coding part of the cDNA for bovine liver-type fatty acid binding protein (L-FABP) has been amplified by RT-PCR, cloned and used for the construction of an Escherichia coli (E. coli) expression system. The recombinant protein made up to 25% of the soluble E. coli proteins and could be isolated by a simple two step protocol combining ion exchange chromatography and gel filtration. Dissociation constants for binding of oleic acid, arachidonic acid, oleoyl-CoA, lysophosphatidic acid and the peroxisomal proliferator bezafibrate to L-FABP have been determined by titration calorimetry. All ligands were bound in a 2:1 stoichiometry, the dissociation constants for the first ligand bound were all in the micro molar range. Oleic acid was bound with the highest affinity and a Kd of 0.26 microM. Furthermore, binding of cholesterol to L-FABP was investigated with the Lipidex assay, a liposome binding assay and a fluorescence displacement assay. In none of the assays binding of cholesterol to L-FABP was observed.
Subject(s)
Carrier Proteins/metabolism , Fatty Acids/metabolism , Myelin P2 Protein/metabolism , Neoplasm Proteins , Amino Acid Sequence , Animals , Base Sequence , Calorimetry , Carrier Proteins/chemistry , Carrier Proteins/genetics , Cattle , Cholesterol/metabolism , Cloning, Molecular , Dextrans , Fatty Acid-Binding Proteins , Fatty Acids/chemistry , Fluorescence , Ligands , Liposomes/chemistry , Liver/metabolism , Lysophospholipids/metabolism , Molecular Sequence Data , Myelin P2 Protein/chemistry , Myelin P2 Protein/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Analysis , ThermodynamicsABSTRACT
An expression vector for bovine heart fatty acid-binding protein (H-FABP) was constructed by introducing the coding part of the cDNA into the pET-3d vector. Transformed Escherichia coli strain BL21 (DE3)pLysS produced functional recombinant H-FABP up to 40% of the soluble proteins. The expression of fatty acid-binding protein was under the control of the T7-phi 10 promoter and the corresponding T7-RNA-polymerase in turn was induced by isopropyl beta-D-thiogalactopyranoside. By combination of cation exchange chromatography and gel filtration pure recombinant protein was obtained exhibiting isoelectric heterogeneity. Recombinant H-FABP was resolved into at least six variants with isoelectric points between 5.1 and 5.6. After separation by preparative isoelectric focusing the four major variants were digested with trypsin and the resulting peptides were characterized by high performance liquid chromatography (HPLC), matrix assisted laser desorption/ionization (MALDI) mass spectrometry, amino acid sequencing and chemical modification. The structural differences were traced back to the N-termini beginning with either methionine, as expected from the cDNA, or methionine sulfoxide, valine and N-formyl methionine. The latter three arise from oxidation, cleavage of N-terminal methionine and incomplete deformylation, respectively.
