In Cellulo Protein Semi‐Synthesis from Endogenous and Exogenous Fragments Using the Ultra‐Fast Split Gp41‐1 Intein

Protein semi‐synthesis inside live cells from exogenous and endogenous parts offers unique possibilities for studying proteins in their native context. Split‐intein‐mediated protein trans‐splicing is predestined for such endeavors and has seen some successes, but a much larger variety of established split inteins and associated protocols is urgently needed. We characterized the association and splicing parameters of the Gp41‐1 split intein, which favorably revealed a nanomolar affinity between the intein fragments combined with the exceptionally fast splicing rate. Following bead‐loading of a chemically modified intein fragment precursor into live mammalian cells, we fluorescently labeled target proteins on their N‐ and C‐termini with short peptide tags, thus ensuring minimal perturbation of their structure and function. In combination with a nuclear‐entrapment strategy to minimize cytosolic fluorescence background, we applied our technique for super‐resolution imaging and single‐particle tracking of the outer mitochondrial protein Tom20 in HeLa cells. To chemically modify proteins inside cells with an exogenously prepared, labeled backbone fragment, we have explored the fastest splicing split intein. Excess intein reagent is removed to the nucleus to increase the signal‐to‐noise ratio in the cytoplasm. We report synthetic fluorophore attachment for super‐resolution and single‐molecule tracking studies.