Development Of Enzymatic Assays Of Sirt1/2/3,And Thereof Activity Evaluation Of The Related Compound

ObjectiveTo construct prokaryotic expression vector pET28a(+)-SIRT1/2/3 and transform it into E.Coli BL21(DE3), and to induce the expression of the SIRT protein. Preparation of the SIRT1/2/3 recombinant proteins, and investigate its enzymology properties and screening of actives compounds. Methods 1. Construction and identification of SIRT1/2/3 expressing plasmidThe DNA encoding SIRT1/2/3 were required by PCR using high-fidelity DNA polymerase. The cloning gene fragment and vector pET28a(+) were digested by restriction enzymes Bam HI and Not I, respectively. Fragements of SIRT1/2/3 DNA and digested vector pET28a(+) were recovered respectively and then linked together to construct prokaryotic expression vector pET28a(+)-SIRT1, pET28a(+)-SIRT2 and pET28a(+)-SIRT3. The SIRT1/2/3 protein were designed to contain single 6-His tag at its N-terminal for purification. The vector pET28a(+)-SIRT1/2/3 were transformed in E. Coli JM109, then the positive clone was screened by kanamycin(50 ?g/mL). The recombinant plasmid was verified by PCR and DNA sequencing. 2. Expression and purification of SIRT1/2/3 recombinant proteinsE.coli cells transformed with the recombinant plasmid were grown in 500 mL Luria Broth medium with kanamycin at 37°C. When OD600 of the culture reached 0.4-0.8, 1 mM IPTG was added to induce bacterial cells to produce recombinant proteins. After incubated at 16°C for overnight, the cells were harvested by centrifugation at 4000 g for 20 minutes. The pellet was stored in-20°C at last for overnight and then suspended in 30 mL of Tris-HCl buffer and disrupted by sonication. After centrifugation to remove the sediment, the supernate was filtered by 0.45?m membrane and purified by affinity chromatography on the Ni-NTA column. The protein supernate was added into Ni-NTA column and then treated with washing buffer(containing 0 mM, 20 mM, 40 mM, 100 mM, 200 mM imidazole) to obtain the target protein, and then analyzed by SDS-PAGE. 3. Development of enzymatic assays of SIRT1/2/3Selected a small peptides near the enzyme catalytic site from the SIRT1/2/3 catalytic substrate proteins, and connect it with fluorescence small molecular(eg. AMC). In the case of connected with peptides, fluorescent molecules cannot be excited fluorescence. But while after the acetyl groups on the lysine residues were transferred by SIRT protein, fluorescent molecules can be released by trypsin shearing, which is triggered release the fluorescence. The substrate peptides used in this research subject derived from p53 protein and its sequence is: Ac- Arg- His- Lys- Lys-(Ac)- AMC. 4. Evaluation method of enzyme activityThe catalytic reaction was performed in the 60 ?L reaction system, 37°C placed 1h later, to add to 60 ?L sample conditioning fluid into the reaction solution, 37°C for 20 minutes. Absorption intensity was measured with a test ex355 em460 using enzyme standard instrument. Results 1. Construction of prokaryotic expression vector pET28a(+)-SIRT1, pET28a(+)-SIRT2 and pET28a(+)-SIRT3The nucleotide sequence of SIRT1, SIRT2 and SIRT3 were confirmed by DNA sequencing, which included 6×His sequence. The recombinant expression vectors were transfected in E.Coli JM109 and positive colonies were screened by kanamycin. The recombinant expression vectors were assayed by PCR. And agarose electrophoresis analysis, special target bands found, suggested that the vector pET28a-SIRT1, pET28a-SIRT2 and pET28a-SIRT3 were constructed successfully. 2. Preparation of recombinant SIRT1/2/3 proteinsThe eluate of 0-buffer, 20 mM, 50 mM, 100 mM, 200 mM buffer were analyzed by SDS-PAGE. The results showed that the eluates of 20 mM was impurity proteins, eluates of 50 mM buffer, 100 mM buffer and 200 mM buffer were target proteins, and the protein content in 100 mM and 200 mM buffer was higher than in the 50 mM buffer. Finally after the desalination and ultrafiltration, the purified recombinant SIRT1/2/3 proteins could be obtained. 3. Activity assay of SIRT1/2/3 proteinsThe determination of enzyme activity of SIRT1/2/3, add different concentration of substrate peptides and NAD+ in the established system of enzyme reaction respectively, by measuring fluorescence intensity to calculate the Km value of SIRT1/2/3 protein. 4. The activity evaluation of related compoundNatural products, resveratrol, was reported to have the activation of SIRT1 activity, but when it comes to the same family SIRT2/3, it has no activation activity. We measured the activity of resveratrol on SIRT1/2/3 to verify the determination method of enzyme activity which we established. Conclusions 1. pET28a-SIRT1, pET28a-SIRT2 and pET28a-SIRT3 recombinant plasmids were constructed successfully. 2. The recombinant expression vectors were transfected into E.coli BL21(DE3), and the purified recombinant proteins were prepared successfully. 3. The enzyme assays were developed, which is enable for evaluation the activities of resveratrol.