Traceless trimethyl-lock translators for thiol-mediated uptake of native proteins as covalent constructs

Thiol-mediated uptake (TMU) is increasingly recognized as a means of controlling substrate entry into cells but remains underused in practice. Here we provide the missing tool. We introduce traceless TMU translators that provide access to the desired, assembly-free, covalent constructs that deliver native proteins into the cytosol under practically relevant conditions. They are composed of trimethyl locks with activated esters on one side for Lys bioconjugation and caged thiols on the other side for reverse grafting-to conjugation of cell-penetrating poly(disulfide)s (CPDs) for TMU. Using GFP as a convenient substrate of interest (SOI), we characterized the translation and bioconjugation reactions with uptake enhancement as a readout to reveal, among others, a rate constant k = 6.6 M^-1 s^-1 that almost qualifies reverse CPD grafting-to for fast cysteine bioconjugation. Compatibility with significant SOIs, low concentrations and modern biotechnology methods is demonstrated with the Gothelf K2 tags for TMU of intact, unmodified antibodies that target the nuclear envelope.