Cytoplasmic versus periplasmic expression of site-specifically and bioorthogonally functionalized nanobodies using expressed protein ligation

- It was proven that it is possible to site-specifically mono-alkynate NbBcII10 using Expressed Protein Ligation.
- With respect to the periplasmic expression of the Nb-intein-CBD complex, several leader sequences were evaluated but none of them were successful.
- By using SHuffle®T7 cells and appending a small peptide spacer (LEY) to the C-terminus of the Nb, fully functional Nb-LEY-alkyne was produced through cytoplasmic expression and alkynation by EPL.
- Nb-intein-CBD fusion complexes, prepared for alkynation by EPL, do not necessarily need to be expressed in the periplasm to remain active and t post-translation alkynation using EPL has no influence on the binding activity of NbBcII10.

Site-specific functionalization of nanobodies after introducing bioorthogonal groups offers the possibility to biofunctionalize surfaces with a uniformly oriented layer of nanobodies. In this paper, expressed protein ligation (EPL) was used for site-specific alkynation of the model nanobody NbBcII10. In contrast to EPL constructs, which are typically expressed in the cytoplasm, nanobodies are expressed in the periplasm where its oxidizing environment ensures a correct folding and disulfide bond formation. Different pathways were explored to express the EPL constructs in the periplasm but simultaneously, the effect of cytoplasmic expression on the functionality of NbBcII10 was also evaluated. By using Escherichia coli SHuffle®T7 cells, it was demonstrated that expression of the EPL complex in the cytoplasm was readily established and that site-specifically mono-alkynated nanobodies can be produced with the same binding properties as the non-modified NbBcII10 expressed in the periplasm. In conclusion, this paper shows that periplasmic expression of the EPL complex is quite challenging, but cytoplasmic expression has proven to be a valuable alternative.