A mesophilic cysteine-less split intein for protein trans -splicing applications under oxidizing conditions

Significance Protein trans -splicing catalyzed by split inteins is a powerful tool for traceless posttranslational protein modification. For example, it can be used to reconstitute a protein from 2 fragments, append a peptide tag to a protein, or cyclize a polypeptide. We report the first cysteine-less (CL) split intein that is high yielding under native and physiological conditions. In contrast to cysteine-containing inteins, the split CL intein is fully operational under oxidizing conditions, i.e., in the absence of reducing agents. Leveraging this property, we utilize the CL intein to selectively conjugate fluorophores to disulfide-containing antibody fragments, and to target membrane receptors on living cells. Carrying out the experimental protocol under oxidizing conditions avoids protein damage due to reduction of disulfide bonds.