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ACS Synth Biol. 2017 Jun 16;6(6):957-967. doi: 10.1021/acssynbio.6b00292. Epub 2017 Mar 7.

Extracellular Self-Assembly of Functional and Tunable Protein Conjugates from Bacillus subtilis.

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Centre for Synthetic Biology and Innovation, Imperial College London , London SW7 2AZ, U.K.
Department of Bioengineering, Imperial College London , London SW7 2AZ, U.K.
Department of Biochemistry, University of Oxford , South Parks Road, Oxford OX1 3QU, U.K.
Centre for Bacterial Cell Biology, Baddiley-Clark Building, Newcastle University , Richardson Road, Newcastle upon Tyne NE2 4AX, U.K.


The ability to stably and specifically conjugate recombinant proteins to one another is a powerful approach for engineering multifunctional enzymes, protein therapeutics, and novel biological materials. While many of these applications have been illustrated through in vitro and in vivo intracellular protein conjugation methods, extracellular self-assembly of protein conjugates offers unique advantages: simplifying purification, reducing toxicity and burden, and enabling tunability. Exploiting the recently described SpyTag-SpyCatcher system, we describe here how enzymes and structural proteins can be genetically encoded to covalently conjugate in culture media following programmable secretion from Bacillus subtilis. Using this approach, we demonstrate how self-conjugation of a secreted industrial enzyme, XynA, dramatically increases its resilience to boiling, and we show that cellular consortia can be engineered to self-assemble functional protein-protein conjugates with tunable composition. This novel genetically encoded modular system provides a flexible strategy for protein conjugation harnessing the substantial advantages of extracellular self-assembly.


SpyTag-SpyCatcher; extracellular self-assembly; microbial consortia; protein conjugation; thermotolerance; tunability

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