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SpyTag is a peptide that spontaneously forms an amide bond with its protein partner SpyCatcher. SpyTag was fused at the N terminus of β-lactamase and SpyCatcher at the C terminus so that the partners could react to lock together the termini of the enzyme. The wild-type enzyme aggregates above 37 °C, with irreversible loss of activity. Cyclized β-lactamase was soluble even after heating at 100 °C; after cooling, the catalytic activity was restored. SpyTag/SpyCatcher cyclization led to a much larger increase in stability than that achieved through point mutation or alternative approaches to cyclization. Cyclized dihydrofolate reductase was similarly resilient. Analyzing unfolding through calorimetry indicated that cyclization did not increase the unfolding temperature but rather facilitated refolding after thermal stress. SpyTag/SpyCatcher sandwiching represents a simple and efficient route to enzyme cyclization, with potential to greatly enhance the robustness of biocatalysts.

Original publication

DOI

10.1002/anie.201402519

Type

Journal article

Journal

Angew Chem Int Ed Engl

Publication Date

10/06/2014

Volume

53

Pages

6101 - 6104

Keywords

enzymes, peptides, protein engineering, protein modifications, protein stability, Cyclization, Models, Molecular, Peptides, Transition Temperature, beta-Lactamases