title: Programmable polyproteams built using twin peptide superglues.
abstract: Programmed connection of amino acids or nucleotides into chains introduced a revolution in control of biological function. Reacting proteins together is more complex because of the number of reactive groups and delicate stability. Here we achieved sequence-programmed irreversible connection of protein units, forming polyprotein teams by sequential amidation and transamidation. SpyTag peptide is engineered to spontaneously form an isopeptide bond with SpyCatcher protein. By engineering the adhesin RrgA from Streptococcus pneumoniae, we developed the peptide SnoopTag, which formed a spontaneous isopeptide bond to its protein partner SnoopCatcher with >99% yield and no cross-reaction to SpyTag/SpyCatcher. Solid-phase attachment followed by sequential SpyTag or SnoopTag reaction between building-blocks enabled iterative extension. Linear, branched, and combinatorial polyproteins were synthesized, identifying optimal combinations of ligands against death receptors and growth factor receptors for cancer cell death signal activation. This simple and modular route to programmable "polyproteams" should enable exploration of a new area of biological space.
authors.affiliation: Department of Biochemistry, University of Oxford, Oxford, OX1 3QU, United Kingdom;
authors.forename: Gianluca
authors.initials: G
authors.lastname: Veggiani
authors.affiliation: Department of Biochemistry, University of Oxford, Oxford, OX1 3QU, United Kingdom; LOC Development Department, R&D Division, Medical Business Unit, Sony Corporation, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan;
authors.forename: Tomohiko
authors.initials: T
authors.lastname: Nakamura
authors.affiliation: Department of Biochemistry, University of Oxford, Oxford, OX1 3QU, United Kingdom;
authors.forename: Michael D
authors.initials: MD
authors.lastname: Brenner
authors.affiliation: Department of Biochemistry, University of Oxford, Oxford, OX1 3QU, United Kingdom; Ecole Normale Supérieure de Lyon, 69364 Lyon Cedex 07, France;
authors.forename: Raphaël V
authors.identifier: http://orcid.org/0000-0002-7031-7228
authors.initials: RV
authors.lastname: Gayet
authors.affiliation: Department of Chemistry, Physical and Theoretical Chemistry Laboratory, University of Oxford, Oxford, OX1 3QZ, United Kingdom.
authors.forename: Jun
authors.initials: J
authors.lastname: Yan
authors.affiliation: Department of Chemistry, Physical and Theoretical Chemistry Laboratory, University of Oxford, Oxford, OX1 3QZ, United Kingdom.
authors.forename: Carol V
authors.initials: CV
authors.lastname: Robinson
authors.affiliation: Department of Biochemistry, University of Oxford, Oxford, OX1 3QU, United Kingdom; mark.howarth@bioch.ox.ac.uk.
authors.forename: Mark
authors.initials: M
authors.lastname: Howarth
mesh_terms: D018829:Adhesins, Bacterial / Q000737:chemistry*; D000269:Adhesives; D004591:Electrophoresis, Polyacrylamide Gel; D010455:Peptides / Q000737:chemistry*; D015398:Signal Transduction; D013296:Streptococcus pneumoniae / Q000737:chemistry
chemical_list: D018829:Adhesins, Bacterial; D000269:Adhesives; D010455:Peptides
keywords: antibody; nanobiotechnology; protein engineering; split protein; synthetic biology