title: Extracellular Self-Assembly of Functional and Tunable Protein Conjugates from Bacillus subtilis.
abstract: 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.
authors.affiliation: 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.
authors.forename: Charlie
authors.initials: C
authors.lastname: Gilbert
authors.affiliation: Department of Biochemistry, University of Oxford , South Parks Road, Oxford OX1 3QU, U.K.
authors.forename: Mark
authors.initials: M
authors.lastname: Howarth
authors.affiliation: Centre for Bacterial Cell Biology, Baddiley-Clark Building, Newcastle University , Richardson Road, Newcastle upon Tyne NE2 4AX, U.K.
authors.forename: Colin R
authors.initials: CR
authors.lastname: Harwood
authors.affiliation: 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.
authors.forename: Tom
authors.identifier: 0000-0001-5392-976X
authors.initials: T
authors.lastname: Ellis
mesh_terms: D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005110:Extracellular Space / Q000378:metabolism*; D006358:Hot Temperature; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D058615:Synthetic Biology / Q000379:methods*
chemical_list: D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D043364:Endo-1,4-beta Xylanases
keywords: SpyTag-SpyCatcher; extracellular self-assembly; microbial consortia; protein conjugation; thermotolerance; tunability