Cloning, Expression, Purification, and Activity Assay of Acetyl-CoA Synthetase from Dunaliella tertiolecta
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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 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 mmol/L, 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/mg.
