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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. Split intein-mediated protein trans-splicing has found extensive applications in chemical biology, protein chemistry, and biotechnology. However, an enduring limitation of all well-established split inteins has been the requirement to carry out the reaction in a reducing environment due to the presence of 1 or 2 catalytic cysteines that need to be in a reduced state for splicing to occur. The concomitant exposure of the fused proteins to reducing agents severely limits the scope of protein trans-splicing by excluding proteins sensitive to reducing conditions, such as those containing critical disulfide bonds. Here we report the discovery, characterization, and engineering of a completely cysteine-less split intein (CL intein) that is capable of efficient trans-splicing at ambient temperatures, without a denaturation step, and in the absence of reducing agents. We demonstrate its utility for the site-specific chemical modification of nanobodies and an antibody Fc fragment by N- and C-terminal trans-splicing with short peptide tags (CysTag) that consist of only a few amino acids and have been prelabeled on a single cysteine using classical cysteine bioconjugation. We also synthesized the short N-terminal fragment of the atypically split CL intein by solid-phase peptide synthesis. Furthermore, using the CL intein in combination with a nanobody–epitope pair as a high-affinity mediator, we showed chemical labeling of the extracellular domain of a cell surface receptor on living mammalian cells with a short CysTag containing a synthetic fluorophore. The CL intein thus greatly expands the scope of applications for protein trans-splicing.