Escherichia coli bl21 de3 cells

Total RNA was extracted using an RNA prep Pure Tissue Kit (Nanjing Vazyme Biotech, Nanjing, China). First-strand cDNA was synthesized using a reverse transcription system kit (Nanjing Vazyme Biotech). The full coding sequence of the non-membrane region of EgHCDH was amplified using the primers 5′- CGG GAT CCA TGT CAG CCG GTG CTG G-3′ (BamHI) and 5′-GAC GTC GAC TCA CTG TTT TTC CTT GAC AAT GCG C-3′ (SalI). Amplification reactions were performed using the following cycling conditions: pre-denaturation at 95 °C, 5 min; then denaturation at 95 °C/30 s, 62 °C/30 s, 72 °C/1 min; with a final extension at 72 °C, 5 min. Through sequencing, digestion and ligation, EgHCDH was ligated into the pET32a (+) plasmid (Novagen, Darmstadt, Germany) and transformed into Escherichia coli BL21 (DE3) cells (Tiangen, Beijing, China). Protein expression was induced with 1 mM isopropyl-1-thio-β-d-galactopyranoside at 37 °C for 6 h. The rEgHCDH protein was purified using Ni²⁺ affinity chromatography (Bio-Rad, Hercules, CA, USA), with the the purity of the final product determined by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The concentrations of the purified protein were determined using a NanoDrop 2000c spectrophotometer (Bio-Rad).

Upstream and downstream oligonucleotides, 5′-CCGGAATTCATAGTCGCTCTAGGCCATTC-3′ and 5′-CCCAAGCTTTCAGTTAAACTTCTTTCGGTG-3′ (EcoRI and HindIII sites are underlined, resp.), were used to amplify the partial Sjlgl coding sequence (GenBank accession number AY812588) from bp 1866–2711. The 846 bp fragment was generated by polymerase chain reaction (PCR) and cloned into the pET-28a (+) vector (Novagen, Darmstadt, Germany), and its identity was confirmed by sequencing.
Overexpression of rSjLGL, with an aminoterminal histidine tag, in Escherichia coli BL21 (DE3) cells (Tiangen Biotech Co., Ltd., Beijing, China) was induced by treatment with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) at 37°C for 5 h. Bacteria were harvested by centrifugation at 10,000 ×g for 15 min. The supernatant was discarded, and the pellet was suspended in 50 mL of phosphate-buffered saline (PBS, pH 7.4) and lysed using ultrasound to release the fusion protein. Following centrifugation at 10,000 ×g for 15 min, the inclusion body protein was extracted in PBS containing urea and purified by passage through Ni-NTA His-Bind Resin (Qiagen GmbH, Hilden, Germany).

The purified recombinant plasmid, pMD19-T-SjVAMP2 was digested with EcoRI and XhoI, and then was ligated to the pET28a (+) vector (Invitrogen) overnight at 16°C using T4 DNA ligase (TaKaRa). The resulting plasmid, pET28a(+)/SjVAMP2, in which a His tag was fused to SjVAMP2, was transformed into Escherichia coli BL21(DE3) cells (Tiangen). Transformed bacterial cells were induced by isopropyl-1-thio-b-D-galactoside (IPTG) to a final concentration of 1 mM at 20°C for 6h, then pelleted by centrifugation at 12,000 g for 15 min, resuspended in phosphate-buffered saline (PBS, pH 7.4), and frozen at −80°C until used. Frozen cells were lysed by sonication and pelleted by centrifugation at 4°C, 12,000 g for 15 min. The insoluble pellet was resuspended in binding buffer containing 8 M urea, while the soluble supernatant was conserved. The crude extract was analyzed by SDS–PAGE.
The supernatant containing rSjVAMP2-His was purified with an Ni-NTA His-Bind Resin (Qiagen GmbH, Hilden, Germany) and dialyzed against PBS (pH 7.4), containing decreasing concentrations of urea (6, 4, 3, 2, and 1 M) and then PBS only. rSjVAMP2-His was concentrated using Centricon microconcentrators (Amicon Millipore, Shanghai, China), and the protein concentration was determined by the Bradford method using bovine serum albumen (BSA) as a protein standard.