New route to protein-coupling via intein trans-splicing in the absence of reducing agents

In protein trans-splicing two parts of a split-intein can be used to ligate or cyclize polypeptides in a traceless manner. One can couple two large molecules, such as proteins to each other (example B on the figure) or couple a large molecule such as a protein or antibody with a marker (example A on the figure). Application for A is, for example, a tumor marker that is delegated to the site of interest with the companion antibody. The intein thereby acts as a catalyst of the coupling reaction and separates (biologically=splices) again from the product, so that the final product has only the two desired parts. The reaction is robust, can be performed in-vivo or in-vitro, using biologically or chemically synthesized peptides or proteins. The sequence of only 2-3 flanking amino acids are constrained. As shown in the figure below, the N and C terminal intein fragments (IntN & IntC) associate and fold into the active domain thereby linking the flanking sequences with a peptide bond. All previously known split inteins only function in reducing conditions due to their use of one or two catalytic cysteines. A German university invented novel cysteine-less split inteins which are capable of robust trans-splicing at ambient temperatures and without the requirement of any chemical reducing or denaturation steps. This allows the preservation of disulfide bonds within the target protein. The use of this split intein was demonstrated for a full-length IgG, an Fc fragment of an IgG antibody and two nanobodies as representatives of therapeutically relevant proteins. The reactions are high yielding (> 90%) at low to medium micromolar concentrations. The university offers a license agreement to companies in the biotechnological or pharmaceutical industry who will make use of the invention in their processes. A research cooperation agreement with industrial partners is also conceivable in order to further develop the invention on the basis of the partner's target protein or antibodies and the intein provided by the university.