Peptide padlocks evolved towards infinite affinity.. (PeptidePadlock)
Peptide padlocks evolved towards infinite affinity for antibody nanoassembly and ultrasensitive cell capture
Start date: May 1, 2014,
End date: Apr 30, 2019
Our ability to tailor individual proteins is now sophisticated, but our ability to assemble such proteins into larger structures is still primitive. Proteins are typically joined by reversible or non-specific linkages. We have designed a unique way to connect protein building blocks irreversibly and precisely, via spontaneous isopeptide bond formation. This involves modifying proteins with a short peptide tag (SpyTag) that is based upon remarkable chemistry used by pathogenic Gram-positive bacteria. Here we will develop this novel approach to address major challenges in synthetic biology. We will engineer SpyTag capture towards infinite affinity (defined as diffusion-limited on-rate and no off-rate), to transform the sensitivity of peptide detection in living systems. We will also apply SpyTag to create a new generation of protein polymers, irreversibly assembled with molecular precision and tailored branching. In parallel we will harness SpyTag to enhance circulating tumor cell (CTC) capture, one of the most promising ways to achieve early cancer diagnosis. In capturing CTCs and other rare cells from blood, the high forces mean that even the strongest non-covalent linkages fail. SpyTag covalent bridging, in concert with super-resolution live cell fluorescence microscopy, will give us the opportunity to answer key questions about the forces and membrane dynamics at the magnetic bead:cell synapse. We will exploit these insights and SpyTag-assembled antibody polymers to dramatically reduce the threshold of antigen expression for CTC capture. This comprehensive program of research will explore novel concepts in protein recognition and cellular response to force, while creating conceptually new tools, making it possible for biologists in a wide range of areas to step beyond existing barriers.
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