现代食品科技2017，Vol.33Issue(3)：67-73,7.DOI:10.13982/j.mfst.1673-9078.2017.3.011
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
摘要
Abstract
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 (bp) (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 identifies 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 mmol/L,and the molecular weight of the expressed DtACS with Trx-His-tagged fusion protein was about 8.74 ku (6.99 ku+l.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/mg.关键词
乙酰辅酶A合成酶/特氏杜氏藻/基因克隆/纯化/比酶活Key words
acetyl-coenzyme A synthetase/Dunaliella tertiolecta/gene cloning/purification/specific enzyme activity引用本文复制引用
金宏昊,梁明华,姜建国..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[J].现代食品科技,2017,33(3):67-73,7.基金项目
国家自然科学基金项目(31171631) （31171631）
