Figure 1.

Sortase A mediated functionalization of nanobodies and assembly of multimeric nanobody constructs. (A) Sortase A recognizes LPXTG motifs near the C-terminus, cleaves the motif after the threonine and forms a thioester acyl-enzyme intermediate. The C-terminal HIS-tag is cleaved off in the process. The acyl-enzyme intermediate is resolved by the addition of the amine nucleophile DBCO-amine or 3-Azido-1-propanamine, which leads to release of sortase and results in ligation of the molecules to the C-terminus of the nanobody. (B) Purified DBCO and azide functionalized nanobodies are incubated for strain-promoted azide-alkyne click chemistry (SPAAC) that results in C-to-C-terminal nanobody fusion and dimer formation. (C) Nanobody dimers connected via polyethylene glycol (PEG)-linker molecules are generated by click reactions of azide-labeled nanobodies on bis-PEG-DBCO. (D) To expand valency, DBCO-labeled nanobodies are attached to multi-arm PEG-azide molecules. Note that illustrations of constructs and PEG-linkers are not to scale.