Guerra D J, Dziewanowska K, Ohlrogge J B, Beremand P D
Biotechnica Canada, Inc., Calgary, Alberta.
J Biol Chem. 1988 Mar 25;263(9):4386-91.
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.
植物酰基载体蛋白（ACP）在大肠杆菌中的表达水平高于组成型大肠杆菌ACP时，似乎并不会显著改变细菌的生长或脂肪酸代谢。在大肠杆菌中表达的植物ACP含有泛酰巯基乙胺，并且约50%以酰基-ACP的形式存在于体内。我们已经纯化并鉴定了重组菠菜ACP-I。氨基末端氨基酸测序表明其与天然菠菜ACP-I一致，并且没有证据表明存在末端甲硫氨酸或甲酰甲硫氨酸。发现重组ACP-I与针对菠菜ACP-I产生的多克隆抗体在免疫上完全交叉反应。重组ACP-I是大肠杆菌脂肪酸合成的不良底物。相比之下，甘蓝型油菜脂肪酸合成酶对重组ACP和大肠杆菌ACP的反应速率相似。同样，从大肠杆菌中分离的丙二酰辅酶A：酰基载体蛋白转酰基酶只能低效地利用重组ACP-I，而来自甘蓝型油菜的相同酶对大肠杆菌ACP或重组ACP-I的反应同样良好。大肠杆菌酰基-ACP合成酶对重组ACP-I的反应速率比对大肠杆菌ACP的反应速率更高。在大肠杆菌中表达菠菜ACP-I首次提供了大量的植物ACP，这应该有助于对该蛋白进行结构分析以及对植物脂质代谢中许多依赖ACP的反应进行研究。
