Recombinant Protein Expression and Purification

Protein recombination and purification were performed as described previously^{22 (link),38 (link)}. Briefly, recombinant proteins were expressed in E. coli strain BL21 [Transetta (DE3) chemically competent cell (Transgen biotech, CD801)]. In brief, 5 μL Luria-Bertani (LB) culture supplemented with 100 μg/μL ampicillin was inoculated with a single colony at 37 °C. After overnight growth, the culture was diluted 100-fold into 300 mL LB supplemented with 100 μg/mL ampicillin. Protein expression was induced in the presence of 0.4 mM IPTG at 16 °C overnight. Then the cell pellets were collected by centrifugation at 5000 rpm, 4 °C for 10 min and purified recombinant proteins using a His or Flag tag Protein Purification Kit (BeaverBeads™) according to the manufacturer’s instruction.