Recombinant Fibre 1 Protein Expression

The recombinant fibre 1 protein was expressed in Escherichia coli. A gene fragment encoding the full-length fibre 1 protein was cloned into the prokaryotic expression vector, pET48a. The recombinant plasmid was transformed into BL21 (DE3) competent cells (TransGene) for inducible expression. The proteins were purified by Ni Sepharose Excel resin (GE Healthcare), following the manufacturer’s recommended protocol. The fibre 2 protein, the CAR protein, and the ECD, D1, and D2 domains of CAR were transiently expressed by transfecting Expi293F cells (Thermo Fisher Scientific) using Polyfectine (Sigma-Aldrich) [27 (link)]. Cell culture supernatants were harvested at 5 days post-transfection and centrifuged at 10,000 × g for 30 min to remove cell debris. The supernatants were sterile-filtered and purified with a protein A resin (GenScript Company). The expression of recombinant proteins was detected by SDS-PAGE and western blot analysis, using appropriate antibodies.