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Split inteins without active site cysteine

Prof. Henning D. Mootz, Westfaelische Wilhelms-University Muenster; Dr Maniraj Bhagawati, Westfaelische Wilhelms-University Muenster; Tobias Terhorst, Westfaelische Wilhelms-University Muenster; Prof. Shmuel Pietrokovski, Weizmann Institute of Science

PROvendis GmbH


Selective modification of proteins and peptides is an important tool for probing natural systems, creating therapeutic conjugates and generating novel protein constructs. These advances are intimately linked to the development of numerous methodologies for protein-protein coupling. Intein bioconjugation still is the unsurpassed technique for simple, efficient and chemoselective covalent protein modification.

However, in general it is difficult or impossible to preserve disulfide bonds within the target protein or to achieve regioselectivity of cystein bioconjugation. The use of split inteins without active site cystein represents a novel selective cystein bioconjugation technique for highly efficient protein labeling and protein coupeling in a traceless manner.


Inteins are protein domains that auto-catalyze protein-splicing of their flanking regions with a peptide bond. In protein trans-splicing two parts of a split-intein can be used to ligate or cyclize polypeptides in a traceless manner. Protein trans-splicing is of particular interest to facilitate the reconstitution of a protein of interest (POI) from two fragments or to modify a POI by transfer of a synthetic, chemically or isotopically labeled part. In both cases, the split intein precursor proteins can be generated and modified individually before being combined to carry out the protein trans-splicing reaction. The reaction is robust, can be performed in vivo or in vitro, using biologically or chemically synthesized peptides or proteins.

However, all previously known split inteins only function in reducing conditions due to their use of one or two catalytic cysteines. In this invention novel cysteine-less split inteins 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.

Commercial Opportunity

The present invention allows for new protein-coupling strategies in the semisynthetic synthesis of recombinant proteins that require site-selective bioconjugation and benefit from the elimination of reducing conditions, which means a preservation of disulfide bonds on one and free cysteins on the other hand.

Development Status

The selectivity, protein-protein coupling efficiency (> 90%) and robustness (low to medium micromolar concentrations) of split inteins without active site cystein was demonstrated for a full-length IgG, an Fc fragment of an IgG antibody and two nanobodies as representatives of therapeutically relevant proteins.

PROvendis is offering licenses for the invention to interested companies on behalf of the University of Muenster and The Weizmann Institute, Israel. There is also the possibility of collaboration with the Inventors.

Patent Situation

US and EP Priority patent applications have been filed so far.

Further Reading

Bhagawati, M. et al. (2019): A mesophilic cysteine-less split intein for protein trans-splicing applications under oxidizing conditions. Proc. Natl. Acad. Sci. U S A, 116 (44): 22164-22172


Split inteins without active site cysteine