Gene synthesis, expression in Escherichia coli, purification and characterization of the recombinant bovine acyl-CoA-binding protein.
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.
Mandrup
S
S
Institute of Biochemistry, Odense University, Denmark.
Højrup
P
P
Kristiansen
K
K
Knudsen
J
J
D000214:Acyl Coenzyme A / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D002417:Cattle; D003001:Cloning, Molecular / Q000379:methods; D026261:Diazepam Binding Inhibitor; D004926:Escherichia coli / Q000235:genetics*; D064429:Fatty Acid Synthases / Q000378:metabolism; D005813:Genes, Synthetic; D006041:Goats; D008969:Molecular Sequence Data; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D000214:Acyl Coenzyme A; D002352:Carrier Proteins; D026261:Diazepam Binding Inhibitor; D011994:Recombinant Proteins; D064429:Fatty Acid Synthases