The in situ cyclization of proteins (INCYPRO) is a protein engineering technology that increases the durability of proteins and enzymes for biotechnological and biomedical applications.[1] For such applications, it is essential that the used proteins maintain their structural integrity.[2] This is, however, often challenged due to the conditions required for these applications which necessitates protein engineering to stabilize the protein structure.[3] The INCYPRO technology involves the attachment of molecular claps (crosslinks) to a protein, thereby reducing the tendency of the protein to unfold. The resulting INCYPRO-crosslinked proteins are more stable at elevated temperature and in presence of chemical denaturants.
The INCYPRO technology utilizes tris-reactive molecules to crosslink three defined positions within a protein[4] or protein complex.[5] For example, INCYPRO can involve the introduction of three spatially aligned and solvent-accessible cysteines into the protein that are then reacted with a tris-electrophilic agent. The resulting crosslinked proteins or protein complexes have been shown to exhibit increased stability towards thermal and chemical stress and a lower tendency towards aggregation. So far, the melting temperature of proteins was increased by up to 39°C in a single design step.
An early example, involved the stabilization of the transpeptidase Sortase A which resulted in INCYPRO-stabilized variants with activity under elevated temperature and in the presence of guanidinium chloride.[6] INCYPRO has also been applied to stabilize the human adaptor KIX domain utilizing different crosslinker molecules. Here, a dependency of protein stability on the hydrophilicity of the crosslink was observed. In addition, a number of homo-trimeric protein complexes was stabilized including the Pseudomonas fluorescens esterase (PFE) and an Enoyl-CoA hydratase. In these cases, enzyme conjugates with overall bicyclic topology were generated.