Recombinant Nanobody Purification Protocol

The sequences of proteins were designed according to the literature [4 (link),16 (link),36 (link),37 (link)]. Coding sequences were subcloned into the E. coli periplasmic expression vector pET-22b(+) with 6 His-tag in the C-terminal. Rosetta E. coli containing the plasmid were grown at 37 °C in LB plus ampicillin. After inducing with 1 mM IPTG at 30 °C for 16 h, the cells were harvested by centrifugation, resuspended in buffer (50 mM Tris, 300 mM NaCl, pH = 8.0), and lysed by ultrasonic. The periplasmic fraction was isolated by centrifugation at 10,000 g for 20 min at 4 °C and then loaded onto Ni-NTA in buffer (50 mM Tris, 300 mM NaCl, 10 mM imidazole, pH = 8.0). Proteins were eluted by a gradient to 50 mM Tris, 300 mM NaCl, 500 mM imidazole, 10% glycerin, pH = 8.0. The eluted proteins were loaded onto a Superdex S-200 column to increase purity. Recombinant nanobodies were assessed by 15% SDS-PAGE. The purified proteins were stored at −80 °C.
To remove bacterial endotoxin, proteins were immobilized on HisTrap HP 5 mL column (GE Healthcare) in PBS. The proteins were washed with PBS containing 0.1% Triton X-114, and eluted with LPS-free PBS with 500 mM imidazole. Imidazole was removed by dialysis. LPS concentration was measured using an LAL Chromogenic Endotoxin Quantitation Kit (Thermo Fisher Scientific, Waltham, MA, USA), and all proteins were LPS purified (<2 IU/mg).