Transb de3 competent cells

The recombinant pCold-SUMO-LBD1 plasmid was transformed into TransB (DE3) competent cells (TransGen Biotech, China) to induce the expression of His₆-SUMO-LBD1 with 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 18 h at 16°C in the presence of 0.5 mM CaCl₂ in LB medium. His₆-SUMO-LBD1 was purified using prepacked Ni-NTA affinity column (GE Healthcare, USA). Phenylmethanesulfonyl fluoride (PMSF) (Sigma, USA) was applied during the entire purification process to inhibit the protease activity. Purified His₆-SUMO-LBD1 was subsequently desalted with HiPrep 26/10 column (GE Healthcare, USA) to remove extra imidazole. His₆-SUMO tag was separated from LBD1 by Ni-NTA column followed the treatment of SUMO protease (20 U/μL) at 4°C for 16 h. Purified LBD1 was analyzed by 12.5% SDS-PAGE and Superdex 200 (GE Healthcare, USA) to determine its molecular weight and conformation in solution. LBD1 was concentrated via ultrafiltration and its final concentration was determined by BCA kit (Beyotime, China). The truncated mutants were expressed and purified as a similar purification procedure of the wild type LBD1 except His₆-Trx tag of pET-32M∙3C vector was cleaved from LBD1 mutants by PreScission protease (GE Healthcare, USA).

The production of truncated CaHEV-ORF2 protein ap237 (aa313-aa549) has been previously described (19 (link)). Recombinant CaHEV-ORF3 protein (ORF3^CaHEV) fused with a 6×His tag was produced by ligating the cDNA of CaHEV-ORF3 to the pET21b (Omega Bio-Tek, Noclos, GA, USA) vector and transforming it into TransB(DE3) competent cells (TransGen Biotech, Beijing, China). Protein expression was induced with 0.5 M isopropyl-beta-d-thiogalactoside (IPTG, Sigma–Aldrich) at 28°C for 8 h before centrifugation and sonication. Next, the recombinant ORF3^CaHEV protein from the supernatant of the bacterial lysate was purified using Ni²⁺ affinity chromatography (TransGen Biotech) and eluted with elution buffer as previously described (19 (link)). The purified ap237 and ORF3^CaHEV proteins were quantified using a Pierce BCA Protein assay kit (Thermo Fisher Scientific) according to the manufacturer's instructions.