Versatile and Efficient Site‐Specific Protein Functionalization by Tubulin Tyrosine Ligase

Abstract A novel chemoenzymatic approach for simple and fast site‐specific protein labeling is reported. Recombinant tubulin tyrosine ligase (TTL) was repurposed to attach various unnatural tyrosine derivatives as small bioorthogonal handles to proteins containing a short tubulin‐derived recognition sequence (Tub‐tag). This novel strategy enables a broad range of high‐yielding and fast chemoselective C‐terminal protein modifications on isolated proteins or in cell lysates for applications in biochemistry, cell biology, and beyond, as demonstrated by the site‐specific labeling of nanobodies, GFP, and ubiquitin.