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.