Protein trans-splicing applications in mesophilic oxidizing conditions using a novel engineered cysteine-less split intein

Inteins are protein domains that self-excise out of their protein hosts ligating their flanks by a peptide bond. The reaction needs no other protein, co-factor, or energy source, is highly accurate and efficient, and readily functions in-vivo or in-vitro. Protein ligation catalyzed by split inteins is a powerful tool for traceless posttranslational protein modification. It can be used to assemble proteins from fragments, append peptide tags to proteins, or cyclize polypeptides. We identified and further engineered the first cysteine-less (CL) split intein that is high-yielding under native and physiological conditions. Unlike cysteine-containing inteins, the split CL intein is fully functional in oxidizing conditions, i.e., in the absence of reducing agents. We used the CL intein to selectively conjugate fluorophores to disulfide-containing antibody and nanobody fragments, and to target membrane receptors on Human living cells. Carrying out the reactions with no reducing agents avoids protein damage due to reduction of disulfide bonds.