Cytoplasmic Production of Nanobodies and Nanobody-Based Reagents by Co-Expression of Sulfhydryl Oxidase and DsbC Isomerase

Nanobodies are stable molecules that can often fold correctly even in the absence of the disulfide bond(s) that stabilize their three-dimensional conformation. Nevertheless, some nanobodies require the formation of disulfide bonds, and therefore they are commonly expressed from vectors that promote their secretion into the oxidizing environment of the Escherichia coli periplasm. As an alternative, the bacterial cytoplasm can be an effective compartment for producing correctly folded nanobodies when sulfhydryl oxidase and disulfide-bond isomerase activities are co-expressed from a recombinant vector. The larger volume and wider chaperone/foldase availability of the cytoplasm enable the achievement of high yields of both nanobodies and nanobody-tag fusions, independently of their redox requirements. Among other examples, the protocol described here was used to successfully produce nanobody fusions with fluorescent proteins that do not fold correctly in the periplasm, nanobodies with Fc domains, and nanobodies containing free cysteine tags.