Authors
McConnell, Scott; Amer, Brendan; Muroski, John; Fu, Janine; Chang, Chungyu; Ogorzalek Loo, Rachel; Loo, Joseph; Osipiuk, Jerzy; Ton-That, Hung; Clubb, Robert
Abstract
Proteins that are site-specifically modified with peptides and chemicals can be used as novel therapeutics, imaging tools, diagnostic reagents and materials. However, there are few enzyme-catalyzed methods currently available to selectively conjugate peptides to internal sites within proteins. Here we show that a pilus-specific sortase enzyme from Corynebacterium diphtheriae ((Cd)SrtA) can be used to attach a peptide to a protein via a specific lysine-isopeptide bond. Using rational mutagenesis we created (Cd)SrtA(3M), a highly activated cysteine transpeptidase that catalyzes in vitro isopeptide bond formation. (Cd)SrtA(3M) mediates bioconjugation to a specific lysine residue within a fused domain derived from the corynebacterial SpaA protein. Peptide modification yields greater than >95% can be achieved. We demonstrate that (Cd)SrtA(3M) can be used in concert with the Staphylococcus aureus SrtA enzyme, enabling dual, orthogonal protein labeling via lysine-isopeptide and backbone-peptide bonds.