Improvement of Connectase-mediated protein ligations and their application in protein detection

• Applicant: Dr. Adrian Fuchs
• Subject Area: Biochemistry
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 512378754

Connectase is a newly discovered protein ligase, an enzyme that catalyzes the reversible fusion of two molecules via an amino acid recognition sequence. It shows high potential for biotechnological applications, such as the generation of hybrid proteins, the immobilization of target proteins or their conjugation to pharmacophores. However, we are only at the very beginning of unlocking that potential. Both the currently employed Connectase ortholog and the associated methodology offer ample potential for improvement. In the first part of this project, we aim to manipulate the ligase reaction to render it irreversible and to increase fusion product yields. We will also establish a high-throughput activity assay, in order to identify favorable Connectase orthologs for specific conditions (e.g. low temperatures) and to find Connectase orthologs with mutually exclusive recognition motifs. Such variants allow the simultaneous conjugation of two protein substrates with different labeling partners, for example different fluorophores. Compared to other protein ligases, Connectase is far more specific for its substrates and catalyzes protein fusions without side reactions. It is therefore well suited for applications in complex solutions with low concentrations of target protein. In the second part of this project, we plan to use this property to label such target proteins with fluorophores, enabling their detection on polyacrylamide gels, on microwell plates, on the cell surface or within the cytoplasm. To date, such applications rely mainly on the use of labeled (e.g. fluorophore-conjugated) antibodies, some of which are specific for genetically introduced tags in the protein of interest. The use of Connectase allows protein detection via a similar tag (i.e. the recognition sequence), but promises a higher sensitivity with less noise, more reliable quantifications, simpler and faster assay protocols, and better reproducibility.

• DFG Programme: Research Grants