Beremand P D, Hannapel D J, Guerra D J, Kuhn D N, Ohlrogge J B
Arch Biochem Biophys. 1987 Jul;256(1):90-100. doi: 10.1016/0003-9861(87)90428-0.
A synthetic gene of 268 bp encoding the 82 amino acid spinach acyl carrier protein (ACP)-I was constructed based on the known amino acid sequence. Two gene fragments, one encoding the amino-terminal portion and the other the carboxy-terminal portion of the protein, were assembled from synthetic oligonucleotides and inserted into the phage M13mp19. These partial gene constructions were joined and inserted into the plasmid pTZ19R. DNA sequencing confirmed the accuracy of the constructions. The synthetic gene was then subcloned into the Escherichia coli expression vector pKK233-2, under the control of the trc promoter. Western blot analysis and radioimmunoassay indicated that E. coli cells carrying this plasmid produced up to 6 mg/liter of a protein which was immunologically cross-reactive and similar in electrophoretic mobility to authentic spinach acyl carrier protein. The bacterial cells were able to attach the phosphopantetheine prosthetic group to the synthetic plant gene product allowing it to be acylated in vitro by acyl-ACP synthetase.
基于已知的氨基酸序列构建了一个268 bp的合成基因，其编码82个氨基酸的菠菜酰基载体蛋白（ACP）-I。从合成寡核苷酸组装了两个基因片段，一个编码该蛋白的氨基末端部分，另一个编码羧基末端部分，并将其插入噬菌体M13mp19中。将这些部分基因构建体连接并插入质粒pTZ19R中。DNA测序证实了构建体的准确性。然后将合成基因亚克隆到大肠杆菌表达载体pKK233-2中，置于trc启动子的控制下。蛋白质免疫印迹分析和放射免疫测定表明，携带该质粒的大肠杆菌细胞产生了高达6 mg/升的一种蛋白质，该蛋白质具有免疫交叉反应性，并且在电泳迁移率上与天然菠菜酰基载体蛋白相似。细菌细胞能够将磷酸泛酰巯基乙胺辅基连接到合成的植物基因产物上，使其能够在体外被酰基-ACP合成酶酰化。
