特氏杜氏藻中乙酰辅酶A合成酶的基因克隆、表达、纯化及活性测定

Cloning, Expression, Purification, and Activity Assay of Acetyl-CoA Synthetase from Dunaliella tertiolecta

乙酰辅酶A合成酶(ACS)催化合成乙酰辅酶A,它是油脂代谢和醋酸盐代谢的重要节点之一。本研究利用反转录PCR(RT-PCR)技术和cDNA末端快速扩增(RACE)技术分离得到了特氏杜氏藻(Dunaliella tertiolecta)乙酰辅酶A合成酶(Dt ACS)的c DNA全长(2464 bp),预测其开放阅读框(ORF)为2184 bp,727个氨基酸由此段编码。序列比对显示Dt ACS与绿藻ACS最为相似(与衣藻Chlamydomonas reinhardtii有68%一致;与团藻Volvox carteri f.nagariensis有70%一致)。选用带有硫氧还蛋白标签(Trx-tag)的pET32a(+)作为原核表达载体,并将Dt ACS转入pET32a(+)中从而构建了pET-32a-Dt ACS质粒。将其转入BL21(DE3)感受态细胞中,同时将pET-32a空载体也转入BL21(DE3)感受态细胞中,分别得到重组菌pET-32a-Dt ACS-BL21(DE3)和对照菌种pET-32a-BL21(DE3)。将重组菌在18℃、终浓度为0.6 mmol/L的IPTG条件下诱导12 h,表达出来的带有Trx-His标签融合蛋白的Dt ACS约为8.74 ku(6.99ku+1.75 ku)。此外,将表达得到的重组蛋白经Ni2+亲和层析柱纯化,纯化后蛋白比活力为52.87 U/mg。

Acetyl-coenzyme A （CoA） synthetase （ACS） catalyzes the synthesis of acetyl-CoA and is one of the important hubs of fat and acetate metabolism. In this study, reverse-transcription polymerase chain reaction （RT-PCR） and rapid amplification of cDNA ends （RACE） techniques were used to obtain ACS cDNA from Dunaliella tertiolecta （DtACS）. The cDNA contained 2,464 base pairs （10p） （full-length）. The predicted length of open reading frame （ORF） was 2,184 bp, and 727 amino acids were encoded by the ORF. Sequence alignment showed that DtACS shared high identities with ACS from chlorophyta （Chlamydomonas reinhardtii, 68% identity and Volvox carteri f. nagariensis, 70% identity）, pET32a （＋） with the thioredoxin tag （Trx-tag） was selected as the prokaryotic expression vector, and DtACS was cloned into pET32a （＋） to construct pET-32a-DtACS, which was then transformed into an Escherichia coli strain BL21（DE3）. The blank vector pET-32a （＋） was also transformed, successfully producing pET-32a-DtACS-BL21（DE3） and control pET-32a-BL21（DE3） recombinant bacteria. The recombinant bacteria were induced at 18 ℃ for 12 h with a final IPTG concentration of 0.6 mmoFL, and the molecular weight of the expressed DtACS with Trx-His-tagged fusion protein was about 8.74 ku （6.99 ku＋1.75 ku）. In addition, the recombinant protein was purified by nickel ion affinity chromatography column, and the specific enzyme activity of the purified protein was 52.87 U/rag.