Gene synthesis, expression in Escherichia coli, purification and characterization of the recombinant bovine acyl-CoA-binding protein

TY - JOUR

T1 - Gene synthesis, expression in Escherichia coli, purification and characterization of the recombinant bovine acyl-CoA-binding protein

AU - Mandrup, S

AU - Højrup, P

AU - Kristiansen, K

AU - Knudsen, J

N1 - Keywords: Acyl Coenzyme A; Amino Acid Sequence; Animals; Base Sequence; Carrier Proteins; Cattle; Cloning, Molecular; Diazepam Binding Inhibitor; Escherichia coli; Fatty Acid Synthetase Complex; Genes, Synthetic; Goats; Molecular Sequence Data; Plasmids; Recombinant Proteins

PY - 1991

Y1 - 1991

N2 - A synthetic gene encoding the 86 amino acid residues of mature acyl-CoA-binding protein (ACBP), and the initiating methionine was constructed. The synthetic gene was assembled from eight partially overlapping oligonucleotides. Codon usage and nucleotides surrounding the ATG translation-initiation codon were chosen to allow efficient expression in Escherichia coli as well as in yeast. The synthetic gene was inserted into the expression vector pKK223-3 and expressed in E. coli. In maximally induced cultures, recombinant ACBP constitutes 12-15% of total cellular protein. A fraction highly enriched for recombinant ACBP was obtained by extracting induced E. coli cells with 1 M-acetic acid. Recombinant ACBP was purified to homogeneity by successive use of gel-filtration chromatography, ion-exchange chromatography and reverse-phase h.p.l.c. Recombinant ACBP differed from native ACBP by lacking the N-terminal acetyl group. The acyl-CoA-binding characteristics of recombinant ACBP did not differ from those of native ACBP, and the two proteins showed the same ability to induce medium-chain acyl-CoA synthesis by goat mammary-gland fatty acid synthetase. It was concluded that the N-terminal acetyl group is not important for acyl-CoA binding.

AB - A synthetic gene encoding the 86 amino acid residues of mature acyl-CoA-binding protein (ACBP), and the initiating methionine was constructed. The synthetic gene was assembled from eight partially overlapping oligonucleotides. Codon usage and nucleotides surrounding the ATG translation-initiation codon were chosen to allow efficient expression in Escherichia coli as well as in yeast. The synthetic gene was inserted into the expression vector pKK223-3 and expressed in E. coli. In maximally induced cultures, recombinant ACBP constitutes 12-15% of total cellular protein. A fraction highly enriched for recombinant ACBP was obtained by extracting induced E. coli cells with 1 M-acetic acid. Recombinant ACBP was purified to homogeneity by successive use of gel-filtration chromatography, ion-exchange chromatography and reverse-phase h.p.l.c. Recombinant ACBP differed from native ACBP by lacking the N-terminal acetyl group. The acyl-CoA-binding characteristics of recombinant ACBP did not differ from those of native ACBP, and the two proteins showed the same ability to induce medium-chain acyl-CoA synthesis by goat mammary-gland fatty acid synthetase. It was concluded that the N-terminal acetyl group is not important for acyl-CoA binding.

M3 - Journal article

C2 - 2064616

SN - 0264-6021

VL - 276 ( Pt 3)

SP - 817

EP - 823

JO - Biochemical Journal

JF - Biochemical Journal

ER -