Cytoplasmic Expression of Nanobodies with Formylglycine Generating Enzyme Tag and Conversion to a Bio-Orthogonal Aldehyde Group.

Nanobodies (Nbs) can be successfully retrieved following phage, bacterial, yeast, or ribosome display of immune, synthetic, or naïve libraries. However, after panning, multiple individual Nb clones need to be screened and assessed for solubility, antigen specificity, affinity, and potential biological function. Therefore, it is highly desirable to have a convenient expression strategy to obtain sufficient protein for in-depth characterization of the Nbs. The presence of a purification and detection tag, as well as a chemically reactive group to enable simple generation of Nb derivatives, would be of great help in this regard. Here, we provide a general protocol for high yield cytoplasmic expression and purification of formylglycine generating enzyme (FGE)-tagged Nbs. The cysteine within the FGE tag is easily converted to formylglycine by passing the FGE-tag containing Nb over a continuous-flow bio-catalysis system. The aldehyde group within the formylglycine side chain at the C-terminal end of the Nb is suitably located for subsequent bio-orthogonal reactions to fluorescent dyes, biotin, polyethylene glycol, or chromatography resins. We also include methods for production of high yield recombinant FGE, as well as conditions for its immobilization on Sepharose to produce the continuous-flow bio-catalysis system.