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.

SUMO targeted ubiqutin ligases (STUbLs) like RNF4 or Arkadia/RNF111 recognize SUMO chains through multiple SUMO interacting motifs (SIMs). Typically, these are contained in disordered regions of these enzymes and also the individual SUMO domains of SUMO chains move relatively freely. It is assumed that binding the SIM region significantly restricts the conformational freedom of SUMO chains. Here, we present the results of extensive molecular dynamics simulations on the complex formed by the SIM2-SIM3 region of RNF4 and diSUMO3. Though our simulations highlight the importance of typical SIM-SUMO interfaces also in the multivalent situation, we observe that frequently other regions of the peptide than the canonical SIMs establish this interface. This variability regarding the individual interfaces leads to a conformationally highly flexible complex. Even though this is in contrast to previous models of the RNF4 SUMO chain interaction, we demonstrate that our simulations are clearly consistent with previous experimental results.