Cloning And Expression Of Human SIRT1

The Sirtuin family is widespread in living organisms, and is a class III histonedeacetylase that is dependented nicotinamide adenine dinulcleotide NAD+, there areseven memners in sirtuin family that were found so far in Mammal SIRT1~SIRT7,and SIRT1(Silent mating type information regulation2homolog1) has beenresearched deeply. SIRT1catalyses the HDAC reaction in which the removal of theacetyl group from the lysine residues is coupled with the hydrolysis of NAD togenerate nicotinamide, lysine, and O-acetyl-ADP-ribose. SIRT1can deacetylate notonly histones but also non-histones, and then regulate a variety of biological processe,including cell grow, apoptosis and aging, adaptation to calorie restriction, andregulation of gene expression. The fuction of SIRT1in tumorigenesis has beenstudied for many years, but we still can not make a conclution that whether SIRT1acts as a tumor promoter or tumor suppressor. Even though SIRT1still may be usedas a therapeutic target for tumors. This study is to prepare SIRT1with higy hydrolyticactivity and specificity using genetic engineering methods.The correct fragment of human SIRT1(193-747) was obtained by site-directedmutagenesis, and then cloned into the pGEM-T Easy vector. By using DNArecombinant techniques, the correct activated fragment was inserted into expressivevector pET28b, and then transformed into E.coli BL21(DE3). Then the positiveclones were induced to express target protein by IPTG. Fusion protein was purifiedby Ni-NTA agarose affinity chromatography.The plasmid pET28b/SIRT1was successfully constructed and transformed intoE.coli BL21(DE3). E.coli expressing pET28b/SIRT1was grown with shaking at37℃.At OD600≈0.8,0.5mM IPTG was added and cells were cultured at20℃over night.The recombined protein was produced in the form of inclusion body. Recombinantprotein was purified by Ni-NTA agarose affinity chromatography. The puritypET28b/SIRT1of was more than90%. At last, the SIRT1activity was evaluated byfluorecense spectrometer, using Ac-Arg-His-Lys-Lys(Ac)-AMC as a substrate. In conclusion, we have established a prokaryotic expression system to generate purifiedactive SIRT1, which can be used for SIRT1inhibitor and activator screening in vitro.