title: Purification and characterization of recombinant spinach acyl carrier protein I expressed in Escherichia coli.
abstract: Expression of plant acyl carrier protein (ACP) in Escherichia coli at levels above that of constitutive E. coli ACP does not appear to substantially alter bacterial growth or fatty acid metabolism. The plant ACP expressed in E. coli contains pantetheine and approximately 50% is present in vivo as acyl-ACP. We have purified and characterized the recombinant spinach ACP-I. NH2-terminal amino acid sequencing indicated identity to authentic spinach ACP-I, and there was no evidence for terminal methionine or formylmethionine. Recombinant ACP-I was found to completely cross-react immunologically with polyclonal antibody raised to spinach ACP-I. Recombinant ACP-I was a poor substrate for E. coli fatty acid synthesis. In contrast, Brassica napus fatty acid synthetase gave similar reaction rates with both recombinant and E. coli ACP. Similarly, malonyl-coenzyme A:acyl carrier protein transacylase isolated from E. coli was only poorly able to utilize the recombinant ACP-I while the same enzyme from B. napus reacted equally well with either E. coli ACP or recombinant ACP-I. E. coli acyl-ACP synthetase showed a higher reaction rate for recombinant ACP-I than for E. coli ACP. Expression of spinach ACP-I in E. coli provides, for the first time, plant ACP in large quantities and should aid in both structural analysis of this protein and in investigations of the many ACP-dependent reactions of plant lipid metabolism.
authors.affiliation: Biotechnica Canada, Inc., Calgary, Alberta.
authors.forename: D J
authors.initials: DJ
authors.lastname: Guerra
authors.forename: K
authors.initials: K
authors.lastname: Dziewanowska
authors.forename: J B
authors.initials: JB
authors.lastname: Ohlrogge
authors.forename: P D
authors.initials: PD
authors.lastname: Beremand
mesh_terms: D000213:Acyl Carrier Protein / Q000235:genetics* / Q000302:isolation & purification; D002849:Chromatography, Gas; D004926:Escherichia coli / Q000235:genetics*; D010944:Plants / Q000032:analysis / Q000235:genetics*; D011994:Recombinant Proteins / Q000302:isolation & purification*
chemical_list: D000213:Acyl Carrier Protein; D011994:Recombinant Proteins