Superdex s200 16 600 column

For mammalian expression of Ty1-Fc, the sequence encoding the nanobody Ty1 was cloned upstream of a human IgG1-Fc. The plasmid was used to transiently transfect FreeStyle 293F cells using the FreeStyle MAX reagent (Life Technologies, Carlsbad, CA, USA) in accordance with the manufacturer’s instructions. The Ty1-Fc fusion was purified from filtered supernatant on Protein G Sepharose (Cytiva, Uppsala, Sweden), eluted in 0.2 M glycine pH 2.2, and purified by size-exclusion chromatography using a Superdex S200 16/600 column (Cytiva, Uppsala, Sweden) in 50 mM Tris pH 7.5 and 150 mM NaCl.

To generate homo- and heterodimers, the different nanobodies were first site-specifically functionalized on the C-terminus using sortase A with either an azide or a dibenzocyclooctine (DBCO) as described in detail here33. In brief, for functionalization with DBCO, 70 µM of nanobody was incubated with 5 µM sortase A, 8 mM DBCO-amine (Sigma–Aldrich) in 150 mM NaCl, 50 mM Tris pH 7.5 and 10 mM CaCl₂ for 3 h at 25 °C. To functionalize the nanobody with an azide, 70 µM of nanobody, was incubated with 5 µM sortase A 5 M, 10 mM 3-azido-1-propanamine (Sigma–Aldrich #762016) in 150 mM NaCl, 50 mM Tris pH 7.5 and 10 mM CaCl₂ for 3 h at 25 °C. In both reactions, unreacted nanobody, sortase A and excess nucleophile were removed using Ni-NTA resin and PD-10 columns or size-exclusion chromatography. The click reaction was initiated by mixing azide and DBCO labeled nanobody in a 1:1 molar ratio for 48 h at 4 °C. Dimers were purified from unreacted monomeric nanobody by size-exclusion chromatography on a Superdex S200 16/600 column (Cytiva).