Fig. 1

EL2BH isolation and purification by streptavidin-mutein. a C-terminal sequence of EL2BH. b EL2BH from two preparations analyzed in SDS-PAGE and stained with Coomassie blue (left panel) or transferred to a PVDF membrane and further stained with His tag antibody (right panel). c SDS-PAGE of biotinylated nanobody EL2BH affinity purified by streptavidin-mutein using either 0.5 M (lane 1), or 1 M ammonium sulphate (lane 2). At 1 M ammonium sulphate much of the nanobody sticks to the column matrix and can be eluted using biotin and mechanical resuspension of the matrix (lane 3). Upon elution with biotin at 0.5 M ammonium sulphate, no EL2BH remained on the matrix (not shown). d SDS PAGE and western blot from crude periplasmic extracts obtained from 1 mmol/1 IPTG (Promega) induced cultures for 1 h at 36 °C that express both EL2BH and BirA, or EL2BH only. Both strains were subsequently grown in LB medium containing either 50 µM biotin, or no extra biotin. Following IPTG induction of all cultures, and incubation for 1 h at 36 °C, periplasmic extracts were analyzed by SDS-PAGE (left panel), and blotted proteins were visualized by QDot-625-conjugated streptavidin (ThermoFisher) under ultraviolet light (geldocEZ, Biorad) (right panel). The arrow points at the highest biotinylation signal that is obtained by overexpression of BirA and addition of extra biotin in the medium