Xhoi restriction enzymes

Antimicrobial peptide Lc1687 was expressed using a pET-32a vector. Primers Lc1687-F/R (Table S1) with restriction sites were used to amplify the Lc1687 gene. The target PCR fragment was inserted into the expression vector pET-32a with TRX-His-tag after being digested with EcoRI and XhoI restriction enzymes (TaKaRa, Dalian, China). The recombinant plasmid (pET-32a-Lc1687) and the parent vector pET-32a (negative control) were both transformed into E. coli BL21 (DE3) and expressed with IPTG induction at 20 °C for 16 h. The bacteria were harvested by centrifugation at 7,000 r/min for 20 min and resuspended in buffer A (20 mM imidazole, 10 mM Na₂HPO₄, 140 mM NaCl, 1.8 mM KH₂PO₄, 2.7 mM KCl, pH 7.4) for sonication. Afterward, the supernatant was collected by centrifugation at 12,000 g for 20 min and loaded onto a His-tag column. The His-tag column was equilibrated with buffer A, followed by washing with buffer B (40 mM imidazole, pH 7.4) to remove non-target proteins, and, finally, the target protein was collected with elution buffer (500 mM imidazole, pH 7.4). The purified protein was analyzed by 12% SDS-PAGE and dialyzed with PBS buffer (0.01M, pH 7.4) for desalination.

The mTNFα (77-235 aa) DNA sequence was cloned into a pET-28a (+) vector using BamHI and XhoI restriction enzymes (Takara, Beijing, China). The plasmid was transformed into Escherichia coli BL21 (DE3) cells and plated on LB-Agar (1% tryptone, 0.5% yeast extract, 1% NaCl, 1.5% agar) with 30 µg/mL Kanamycin sulfate at 37 °C overnight. Single colonies were selected and cultured in LB media. When OD600 reached 0.4–0.6, the culture was induced with 1 mM IPTG and incubated at 30 °C for 16 h. Cells were harvested and washed twice with a PBS buffer (Sangon, Biotech, Shanghai, China). Then, the cells were lysed by Ultrasonic machining in a lysis buffer (25 mM Tris, 150 mM NaCl, 1 mM PMSF, 1 mg/mL lysozyme, 10% glycerol, pH 7.5). Protein extracts were collected by centrifugation at 12,000 rpm for 30 min. The mTNFα containing His-tag was purified by ProteinIso Ni-NTA resin (TransGen Biotech, Beijing, China) and natively eluted with a different imidazole buffer (25 mM Tris, 150 mM NaCl, 25–500 mM imidazole, 10% glycerol, pH 7.5). The eluted mTNFα protein was subsequently dialyzed with a dialysis buffer (1 × PBS, pH 7.5), and a portion of the mTNFα protein was treated by thrombin protease overnight to remove His-tag.