裂殖壶菌酰基载体蛋白(ACP)基因的克隆与表达

Cloning and Expression of Acyl Carrier Protein Gene from Schizochytrium

摘要:

ACP (Acyl carrier protein, 酰基载体蛋白) 参与高度不饱和脂肪酸的PKS (Polyketide synthase) 生物合成途径。从Schizochytrium sp.FJU-512 cDNA文库中获得了ACP基因的cDNA克隆。该序列开放读码框全长429 bp, 编码142个氨基酸, 等电点为5.04, 具有4′-磷酸泛酰巯基乙胺(4′-PP)的结合位点。利用BamHⅠ/HindⅢ双酶切, 并连接到原核表达载体pET-30a, 构建了pET-30a/acp表达载体, 转化宿主菌E.coli BL21(DE3), IPTG诱导表达。SDS-PAGE分析表明该蛋白得到高效表达。

Abstract:

Acyl carrier protein is an essential component involved in the biosynthesis of DHA (Docosahexaenoic Acid) via PKS (Polyketide synthase) pathway, which takes the growing acyl chain from one enzyme to another. One cDNA clone, with high homology of ACP, was isolated from Schizochytrium sp. FJU-512 cDNA library. The deduced amino acid sequence contained 142 residues with isoelectric point of 5.04 and had the 4′-phosphopantetheine prosthetic(4′-PP) binding site. The target fragment was digested with BamHⅠ/HindⅢand inserted into the expression vector pET-30a resulting in the plasmid pET-30a/acp. The recombinant vector was transformed into E.coli BL21(DE3) and induced by IPTG. SDS-PAGE analysis demonstrated that ACP was effectively expressed.