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-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.
Specht
B
B
Department of Biochemistry, University of Münster, Germany.
Oudenampsen-Krüger
E
E
Ingendoh
A
A
Hillenkamp
F
F
Lezius
A G
AG
Spener
F
F
D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D002417:Cattle; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D050556:Fatty Acid-Binding Proteins; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins; D011994:Recombinant Proteins / Q000096:biosynthesis*
D002352:Carrier Proteins; D050556:Fatty Acid-Binding Proteins; D009363:Neoplasm Proteins; D011994:Recombinant Proteins