With high brightness and photostability, quantum dots (QDs) are potent probes for long-term imaging of dynamic cell surface proteins, but practical methods to covalently label QDs to target proteins for stable imaging are largely lacking. Here, a small covalent-bond forming protein (Covalent-avidin)/peptide pair is introduced, which provides a recombinant protein-based rapid and covalent QD labeling strategy. Covalent-avidin is constructed by optimized fusion of circular permuted monomeric avidin to SpyCatcher, which forms an isopeptide bond with the SpyTag peptide. Covalent-avidin-conjugated QDs allow for strong and irreversible QD labeling to the biotinylated SpyTag-fused adrenergic receptor on live cells in 2 min. In addition, QDs with only a minimum number of conjugated Covalent-avidin show more stable receptor labeling than commercially available streptavidin-conjugated QDs, also with minimal unwanted clustering of labeled receptors. Monomeric Covalent-avidin will be a valuable protein linker for diverse other nanolabeling structures with beneficial properties such as covalent linkages and facile valency control.
Keywords: avidin; covalent label; membrane proteins; protein engineering; quantum dots.
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