title: Protein engineering by highly parallel screening of computationally designed variants.
abstract: Current combinatorial selection strategies for protein engineering have been successful at generating binders against a range of targets; however, the combinatorial nature of the libraries and their vast undersampling of sequence space inherently limit these methods due to the difficulty in finely controlling protein properties of the engineered region. Meanwhile, great advances in computational protein design that can address these issues have largely been underutilized. We describe an integrated approach that computationally designs thousands of individual protein binders for high-throughput synthesis and selection to engineer high-affinity binders. We show that a computationally designed library enriches for tight-binding variants by many orders of magnitude as compared to conventional randomization strategies. We thus demonstrate the feasibility of our approach in a proof-of-concept study and successfully obtain low-nanomolar binders using in vitro and in vivo selection systems.
authors.affiliation: Department of Computer Science, University of Toronto, Toronto, Ontario M5S 3E1, Canada.; Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada.
authors.forename: Mark G F
authors.initials: MG
authors.lastname: Sun
authors.affiliation: Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada.
authors.forename: Moon-Hyeong
authors.initials: MH
authors.lastname: Seo
authors.affiliation: Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada.
authors.forename: Satra
authors.initials: S
authors.lastname: Nim
authors.affiliation: Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada.
authors.forename: Carles
authors.identifier: 0000-0002-7654-4405
authors.initials: C
authors.lastname: Corbi-Verge
authors.affiliation: Department of Computer Science, University of Toronto, Toronto, Ontario M5S 3E1, Canada.; Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada.; Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 3E1, Canada.
authors.forename: Philip M
authors.initials: PM
authors.lastname: Kim
mesh_terms: D000595:Amino Acid Sequence; D002151:Calorimetry; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D019151:Peptide Library; D025341:Principal Component Analysis; D011485:Protein Binding; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism; D043222:Ubiquitin Thiolesterase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
chemical_list: D019151:Peptide Library; D011994:Recombinant Proteins; C408875:USP21 protein, human; D025801:Ubiquitin; D004247:DNA; D043222:Ubiquitin Thiolesterase
keywords: Computational protein design; protein engineering; structural biology