Nanobody 2H10 expression

2H10 encoding DNA was cloned into the vector pAX51 (Lutje Hulsik et al., 2013) and expressed in the E. coli BL21(DE3) strain (ThermoFisher). Bacteria were grown at 37°C to an OD_600nm of 0.7 and induced with 1 mM IPTG at 20°C for 20 hr. After harvesting by centrifugation, bacteria were resuspended in lysis buffer containing 20 mM Hepes pH 7.5 and 100 mM NaCl. Bacteria were lysed by sonication and centrifuged at 48,000 g for 30 min. Cleared supernatant was loaded onto a Protein A sepharose column, washed with lysis buffer and eluted with 0.1 M glycine pH 2.9. Eluted fractions were immediately mixed with 1/5 vol of 1M Tris pH 9.0. 2H10 was then further purified by SEC on a superdex 75 column in PBS buffer. Genes corresponding to mutants of 2H10, 2H10-F (S100d) and 2H10-RKRF (S27R, S30K, S74R, and S100d) were synthesized (Biomatik) and the expressed mutant proteins were purified as described for the wild type. The 2H10 bi-head was purified as described (Lutje Hulsik et al., 2013).