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

S Mandrup, P Højrup, K Kristiansen, J Knudsen

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

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.

Original languageEnglish
JournalBiochemical Journal
Volume276 ( Pt 3)
Pages (from-to)817-823
ISSN0264-6021
DOIs
Publication statusPublished - 15. Jun 1991

Keywords

  • Acyl Coenzyme A/metabolism
  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Carrier Proteins/biosynthesis
  • Cattle
  • Cloning, Molecular/methods
  • Diazepam Binding Inhibitor
  • Escherichia coli/genetics
  • Fatty Acid Synthases/metabolism
  • Genes, Synthetic
  • Goats
  • Molecular Sequence Data
  • Plasmids
  • Recombinant Proteins/biosynthesis

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