title: Engineering High Affinity Protein-Protein Interactions Using a High-Throughput Microcapillary Array Platform.
abstract: Affinity maturation of protein-protein interactions requires iterative rounds of protein library generation and high-throughput screening to identify variants that bind with increased affinity to a target of interest. We recently developed a multipurpose protein engineering platform, termed μSCALE (Microcapillary Single Cell Analysis and Laser Extraction). This technology enables high-throughput screening of libraries of millions of cell-expressing protein variants based on their binding properties or functional activity. Here, we demonstrate the first use of the μSCALE platform for affinity maturation of a protein-protein binding interaction. In this proof-of-concept study, we engineered an extracellular domain of the Axl receptor tyrosine kinase to bind tighter to its ligand Gas6. Within 2 weeks, two iterative rounds of library generation and screening resulted in engineered Axl variants with a 50-fold decrease in kinetic dissociation rate, highlighting the use of μSCALE as a new tool for directed evolution.
authors.affiliation: Department of Bioengineering, ‡Institute for Stem Cell Biology and Regenerative Medicine, §Stanford Photonics Research Center, ∥Chemical Engineering, Stanford University , 450 Serra Mall, Stanford, California 94305, United States.
authors.forename: Sungwon
authors.identifier: 0000-0003-4045-6478
authors.initials: S
authors.lastname: Lim
authors.affiliation: Department of Bioengineering, ‡Institute for Stem Cell Biology and Regenerative Medicine, §Stanford Photonics Research Center, ∥Chemical Engineering, Stanford University , 450 Serra Mall, Stanford, California 94305, United States.
authors.forename: Bob
authors.initials: B
authors.lastname: Chen
authors.affiliation: Department of Bioengineering, ‡Institute for Stem Cell Biology and Regenerative Medicine, §Stanford Photonics Research Center, ∥Chemical Engineering, Stanford University , 450 Serra Mall, Stanford, California 94305, United States.
authors.forename: Mihalis S
authors.initials: MS
authors.lastname: Kariolis
authors.affiliation: Department of Bioengineering, ‡Institute for Stem Cell Biology and Regenerative Medicine, §Stanford Photonics Research Center, ∥Chemical Engineering, Stanford University , 450 Serra Mall, Stanford, California 94305, United States.
authors.forename: Ivan K
authors.initials: IK
authors.lastname: Dimov
authors.affiliation: Department of Bioengineering, ‡Institute for Stem Cell Biology and Regenerative Medicine, §Stanford Photonics Research Center, ∥Chemical Engineering, Stanford University , 450 Serra Mall, Stanford, California 94305, United States.
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Baer
authors.affiliation: Department of Bioengineering, ‡Institute for Stem Cell Biology and Regenerative Medicine, §Stanford Photonics Research Center, ∥Chemical Engineering, Stanford University , 450 Serra Mall, Stanford, California 94305, United States.
authors.forename: Jennifer R
authors.initials: JR
authors.lastname: Cochran
mesh_terms: D011485:Protein Binding; D015202:Protein Engineering; D011506:Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D059010:Single-Cell Analysis
chemical_list: D011506:Proteins