30 kda centrifugal filter

SIRT1 (amino acids 193–747) was produced as reported previously^{74 (link)} and expressed in BL21 DE3 using a pTriEx-based expression vector. Cells were grown in 2XTY broth supplied with 100 µg ml⁻¹ carbenicillin at 37 °C until the optical density reached ~0.6, then induced with isopropyl-β-d-thiogalactoside (1 mM) and ZnSO₄ (0.2 mM). Cells were harvested after 3 h of incubation by centrifugation and then lysed by lysozyme in lysis buffer (50 mM Tris pH 8, 5 mM β-mercaptoethanol and 5% glycerol). His-tagged SIRT1 was then purified using standard immobilized metal affinity chromatography. The eluted protein was then dialysed using a Slide-A-Lyzer dialysis cassette 20K MWCO (Millipore) in dialysis buffer (50 mM Tris pH 8, 150 mM KCl, 5 mM β-mercaptoethanol and 5% glycerol) and then concentrated using a 30 kDa centrifugal filter (Millipore). Purified samples were analysed by SDS–PAGE.

As previously described, the expression plasmids pET-22b-Hc-daf-22-84S (299A/ 349A) were transformed into BL21 (DE3) and induced by 1 mM IPTG at 37°C. Recombinant proteins were purified using Ni-NTA agarose (Qiagen, Shanghai, China) according to manufacturer’s protocol. To refold proteins, the purified eluting solution was added into the lumen space of 30 kDa centrifugal filter (Millipore) and centrifuged at 4°C for 10 min with a speed of 4000 rpm. Then the filtrate was discarded and 0.01 M PBS was added into the filter and centrifugation was repeated under the same condition. This step was repeated for 4–5 times to remove the imidazole. After that, PBS was removed by adding of reaction buffer following the same procedures. The final refolded proteins were kept in reaction buffer and concentrations were measured using the BCA method according to manufacturer’s protocol. Hc-DAf-22 were prepared as control in the subsequent protease activity assay.