Recombinant Protein Purification and Antibody Generation

E. coli BL21 (DE3) was transformed separately with pETTraT, pETmCD46, pETmCD46 mutants, and pET32a (which expresses the Trx tag). The transformants were cultured in LB medium at 37 °C to mid-log phase, and the expression of the recombinant proteins (rTraT, rmCD46, rmCD46 mutants, and rTrx) was induced by adding isopropyl-β-D-thiogalactopyranoside to a final concentration of 1 mM. After growth at 16 °C for an additional 16 h, the cells were harvested by centrifugation, and recombinant proteins were purified using nickel-nitrilotriacetic acid columns (GE Healthcare, Piscataway, NJ, USA) as recommended by the manufacturer. The purified proteins were treated with Triton X-114 to remove endotoxin as reported previously^{58 (link)}. The proteins were dialyzed for 24 h against phosphate-buffered saline (PBS) and concentrated using PEG 20000. The concentrations of the purified proteins were determined using NanoPhotometer (Implen GmbH, Munich, Germany). Mouse antibodies against rTraT and rTrx were prepared as reported previously^{59 (link)} and purified using rProtein G Beads (Solarbio, Beijing, China).