Glutathione beads

CqSIRT1 recombinant protein fused with His tag was expressed from pET-32a (+) in E. coli BL21 (DE3), which were both induced with 0.1 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG) for 20 h at 16 °C. Then, the His-tagged CqSIRT1 recombinant protein was purified with a Ni-NTA affinity column (Smart Lifesciences, Changzhou, China) and washed with washing buffer (50 mM Tris-HCl, 0.5 M NaCl, 25 mM imidazole; pH 8.0). Finally, the purified recombinant protein was collected with an elution buffer (50 mM Tris-HCl, 500 mM NaCl, 250 mM imidazole; pH 8.0) and dialyzed with buffer solution (50 mM Tris, 150 mM NaCl, 5% glycerol). The GST-tagged VP28 recombinant prokaryotic expression vector was constructed previously [14 (link)]. The GST-VP24 and GST-VP26 vectors were generated with PCR and subcloned into a pGEX-4T-1 vector between the BamH I and EcoR I restriction sites. These recombinant proteins were also induced with 0.1 mM IPTG and purified with glutathione beads (Smart Lifesciences, Changzhou, China), which was followed by elution with L-glutathione reduced solution (Solarbio, Beijing, China) (50 mM Tris-HCl, 20 mM L-glutathione reduced, 5 mM DTT; pH = 8.0). These recombinant proteins were stained with Coomassie brilliant blue and analyzed in 12% SDS-PAGE.

For analysis of protein from T. reesei mycelia, the intracellular proteins were extracted as previously described. The His-ACE1^317-463aa protein was purified using High Affinity Ni-NTA Resin (GenScript, Nanjing, China) from the intracellular proteins. The purified His-ACE1^317-463aa was digested by trypsin whereafter the resulting peptides mixture was analyzed using a Dionex Ultimate 3000 RSLCnano system coupled to a Orbitrap Fusion Lumos mass spectrometer. For analysis of protein from E. coli, the GST-ACE1^317-463aa protein was purified using Glutathione Beads (Smart-LifeSciences, China) from E. coli BL21 (DE3). The desalted His-ACE1^317-463aa was digested by trypsin and the peptides were separated using RP-C18 column and subsequently analyzed by Q Exactive HF-X (Thermo Fisher).

The CDS of CiAGL9 was cloned into the pET32a vector to generate CiAGL9-His fusion protein, and the CDS of CiMADS43 was cloned into the pGEX-6p-1 vector to generate the CiMADS43-GST fusion protein. The constructs of CiAGL9-His and CiMADS43-GST were transformed into Escherichia coli BL21 (DE3) strain and induction with 1 mM Isopropyl β-d-thiogalactoside (IPTG) during 16 h at 16 °C. All the cells were harvested by centrifugation at 4000× g for 20 min at 4 °C. Total proteins were extracted with the buffer (pH 7.5) containing 0.7 M sucrose, 0.1 M KCl, 0.5 M Tris-HCl, 50 mM EDTA, 2% (v/v) β-mercaptoethanol, and 100 mM phenylmethylsulfonyl fluoride (PMSF). The protein concentration was determined using the BCA reagent (Aidlab) with bovine serum albumin as a standard. CiAGL9-His were incubated with CiMADS43-GST or GST in binding buffer (400 mM NaCl, 50 mM Tris-HCl, 1 mM PMSF, and 2% (v/v) β-mercaptoethanol, pH 8.0) at 4 °C overnight. Glutathione Beads (Smart-Lifesciences, SA008K, Changzhou, China) were washed five times with the pull-down buffer. The bound proteins were released by adding 2× loading buffer and boiled for 5 min at 95 °C, then resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and detected by the anti-His (TransGen Biotech, HT501-01) and anti-GST antibodies (TransGen Biotech, HT601-01, Nanjing, China), respectively.