Abstract
AbstractSpyTag is a peptide that forms a spontaneous amide bond with its protein partner SpyCatcher. This protein superglue is a broadly useful tool for molecular assembly, locking together biological building blocks efficiently and irreversibly in diverse architectures. We initially developed SpyTag and SpyCatcher by rational design, through splitting a domain from a Gram‐positive bacterial adhesin. In this work, we established a phage‐display platform to select for specific amidation, leading to an order of magnitude acceleration for interaction of the SpyTag002 variant with the SpyCatcher002 variant. We show that the 002 pair bonds rapidly under a wide range of conditions and at either protein terminus. SpyCatcher002 was fused to an intimin derived from enterohemorrhagic Escherichia coli. SpyTag002 reaction enabled specific and covalent decoration of intimin for live cell fluorescent imaging of the dynamics of the bacterial outer membrane as cells divide.
Highlights
SpyTag is a peptide that forms a spontaneous amide bond with its protein partner SpyCatcher
We established a phage-display platform to select for specific amidation, leading to an order of magnitude acceleration for interaction of the SpyTag002 variant with the SpyCatcher002 variant
We show that the 002 pair bonds rapidly under a wide range of conditions and at either protein terminus
Summary
SpyTag is a peptide that forms a spontaneous amide bond with its protein partner SpyCatcher. We established a phage-display platform to select for specific amidation, leading to an order of magnitude acceleration for interaction of the SpyTag002 variant with the SpyCatcher002 variant. Engineering covalent interactions between protein partners brings a range of new opportunities for basic research and synthetic biology.[1] We have developed the use of spontaneous amide bond formation by peptide tags as a simple, specific, and genetically-encoded route to lock protein units together.[2] This technology, the SpyTag/SpyCatcher pair, has been used in diverse applications including biomaterials, next-generation sequencing, enzyme stabilization, and vaccine development.[1a,3] A key limitation has been relatively slow reaction at cellular expression levels.
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