Empowering Site‐Specific Bioconjugations In Vitro and In Vivo: Advances in Sortase Engineering and Sortase‐Mediated Ligation.

Sortase‐mediated ligation (SML) has emerged as a powerful and versatile methodology for site‐specific protein conjugation, functionalization/labeling, immobilization, and design of biohybrid molecules and systems. However, the broader application of SML faces several challenges, such as limited activity and stability, dependence on calcium ions, and reversible reactions caused by nucleophilic side‐products. Over the past decade, protein engineering campaigns and particularly directed evolution, have been extensively employed to overcome sortase limitations, thereby expanding the potential application of SML in multiple directions, including therapeutics, biorthogonal chemistry, biomaterials, and biosensors. This review provides an overview of achieved advancements in sortase engineering and highlights recent progress in utilizing SML in combination with other state‐of‐the‐art chemical and biological methodologies. The aim is to encourage scientists to employ sortases in their conjugation experiments. [ABSTRACT FROM AUTHOR]