Natalie M. Rachel1, Éric Levesque1,3, André B. Charette1,3,4 and Joelle N. Pelletier1,2,3,4.
1Department of Chemistry, Université de Montréal, Montréal, QC.
2Department of Biochemistry, Université de Montréal, Montréal, QC.
3CGCC, the Center in Green Chemistry and Catalysis, Montréal, QC.
4PROTEO, the Québec Network for Protein Function, Structure and Engineering, Québec, QC.
Site-specific modification of peptides and proteins allows us to control their chemical, structural, and functional properties. Biocatalyzed conjugation is a promising alternative to traditional metal-catalyzed conjugation, with enzymes offering high specificity for the biological target and compatible with the required mild reaction conditions. Considering this, the natural capacity for transglutaminases to catalyze protein cross-linking via the formation of isopeptide bonds appealed to us as a starting point to engineer new biotechnology. Transglutaminases have been successfully used to incorporate a variety of chemical probes, fluorescent or otherwise, into peptides, proteins, nucleic acids and even tumor cells. We, and others, have determined that the robust microbial transglutaminase (MTG) can use a wide range of synthetic amine-containing compounds as substrates,1 broadening its scope for the modification of glutamine-containing peptides and proteins. To further expand the applicability and chemical diversity of MTG-catalyzed protein labeling, we examine two approaches. First, we present a detailed characterization of a one-pot chemoenzymatic reaction combining MTG and the copper catalyzed azide-alkyne Huisgen cycloaddition, using both peptide and protein glutamine substrates. Both reactions proved to be compatible under certain conditions only and could result in yields of over 80% under high reaction control. Additionally, we revisited direct MTG-catalyzed protein labeling using a novel tunable, amine-bearing fluorophore. Having these reactions in our arsenal, we developed a platform for engineering new forms of MTG with increased reactivity and specificity towards the native sequence of a model protein substrate, protein Gb1. These findings highlight the potential to use MTG as a tool for peptide and protein modification.
1M. T. Gundersen, J. W. Keillor and J. N. Pelletier. Appl Microbiol Biot, 2014, 98, 219-230.