Functional Study Of SIRT3 Gene In Takifugu Rubripes

BackgroundSirtuins(SIRTs)are NAD(+)-dependent deacetylases that catalyze the hydrolysis of acetyl-lysine residues.They play an important role in many physiological and pathophysiological processes,such as the regulation of lifespan and the prevention of metabolic diseases.The silent information regulator 3(SIRT3)plays an essential role in mitochondrial oxidative metabolism,energy metabolism,signal conditioning and apoptosis and other activities,such as the maintenance of intracellular homeostasis.SIRT3 is also an important regulator of apoptosis,due to its regulation of mitochondrial oxidative stress and biological effects,resistance to apoptosis of nerve cells.In the present study,we cloned a cDNA encoding SIRT3 from torafugu Takifugu rubripes and analyzed its prokaryotic expression for better understanding of molecular mechanisms in the metabolic disorders.ObjectiveObtain the sequence of SIRT3 gene in Takifugu rubripes,and bioinformatics analysis.Construction of prokaryotic expression vector of SIRT3 gene in Takifugu rubripes and expression and purification of protein,discusses it in lipid metabolism inbiological function.Methods1.The ORF of torafugu Takifugu rubripes SIRT3 gene was amplified from total RNA of torafugu liver tissues by reverse transcription-polymerase chain reaction(RT-PCR),and sequence analysis by bioinformatics.2.Construction of prokaryotic expression vector of PET-32/ SIRT3 in Escherichia coli Rosetta(DE3)was expressed after induced by IPTG for expression of fusion protein.The protein was identified by Western blot.Results 1.The cloning and sequence analysis of the torafugu SIRT3The complete open reading frame(ORF)of Takifugu rubripes SIRT3 which obtained using RT-PCR,is 1263 bp in length and encoding a polypeptide of 420 amino acids,the molecular weight is 45 kD and the isoelectric point is 8.10.The multiple sequence alignment showed that coding amino acid sequences of the obtained torafugu SIRT3 gene had 87%,86%,83%,77%,60%,59%,58%,57% homology with the corresponding Larimichthys crocea,Esox lucius,Cynoglossus semilaevis,Oreochromis niloticus,Xenopus laevis,Oryctolagus cuniculus,Heterocephalus glaber respectively.The Phylogenetic tree analysis showed that torafugu SIRT3 is closely related to Larimichthys crocea SIRT3 than to those from other vertebrate species.2.The construction of the torafugu pET-32a(+)/SIRT3Primer design a package of Takifugu rubripes SIRT3 gene ORF region with double enzyme cutting sites,PCR and cloned into pET-32a(+)prokaryotic expression vector,double digestion and sequencing confirmed the success of double identification,the recombinant plasmid pET-32a(+)/SIRT3.3.The expression of the torafugu SIRT3The pET-32a(+)/SIRT3 recombinant vector was transformed into Rosetta(DE3)E coli using 1.0 mM IPTG for induction.The protein size was 66 Das estimated by SDS-PAGE.4.The purification and identification of the pET-32a(+)/SIRT3 of torafuguThe obtained supernatant was purified by 6×His Ni-NTA affinity chromatography.SDS-PAGE and Western blot showed that the molecular weight of the target protein is 66 kD,that purified fusion protein was torafugu SIRT3.ConclusionsWe cloned the full-length cDNA encoding SIRT3 from torafugu liver using RT-PCR,The prokaryotic expression system of recombined vector pET-32a/SIRT3 was construted successfull.The fusion proteins with His tag were also purified and the molecular weight of the recombinant protein was about 66 KD.