N-TERMINAL VARIANTS OF FATTY-ACID-BINDING PROTEIN FROM BOVINE HEART OVEREXPRESSED IN ESCHERICHIA-COLI

An expression vector for bovine heart fatty acid-binding protein (H-FA BP) was constructed by introducing the coding part of the cDNA into th e pET-3d vector. Transformed Escherichia coli strain BL21 (DE3)pLysS p roduced functional recombinant H-FABP up to 40% of the soluble protein s. The expression of fatty acid-binding protein was under the control of the T7-phi 10 promoter and the corresponding T7-RNA-polymerase in t urn was induced by isopropyl beta-D-thiogalactopyranoside. By combinat ion of cation exchange chromatography and gel filtration pure recombin ant protein was obtained exhibiting isoelectric heterogeneity. Recombi nant H-FABP was resolved into at least six variants with isoelectric p oints between 5.1 and 5.6. After separation by preparative isoelectric focusing the four major variants were digested with trypsin and the r esulting peptides were characterized by high performance liquid chroma tography (HPLC), matrix assisted laser desorption/ionization (MALDI) m ass spectrometry, amino acid sequencing and chemical modification. The structural differences were traced back to the N-termini beginning wi th either methionine, as expected from the cDNA, or methionine sulfoxi de, valine and N-formyl methionine. The latter three arise from oxidat ion, cleavage of N-terminal methionine and incomplete deformylation, r espectively.