title: Investigating affinity-maturation strategies and reproducibility of fluorescence-activated cell sorting using a recombinant ADAPT library displayed on staphylococci.
abstract: During the past decades, advances in protein engineering have resulted in the development of variousin vitroselection techniques (e.g. phage display) to facilitate discovery of new and improved proteins. The methods are based on linkage between genotype and phenotype and are often performed in successive rounds of selection. Since the resulting output depends on the selection pressures used and the applied strategy, parameters in each round must be carefully considered. In addition, studies have reported biases that can cause enrichment of unwanted clones and/or low correlation between abundance in output and affinity. We have recently developed a selection method based on display of protein libraries onStaphylococcus carnosusand isolation of affinity proteins by fluorescence-activated cell sorting. Here, we compared duplicate selections for affinity maturation using equilibrium binding at different target concentrations and kinetic off-rate selection. The results showed that kinetic selection is efficient for isolation of high-affinity binders and that equilibrium selection at subnanomolar concentrations should be avoided. Furthermore, the reproducibility of the selection was high and a clear correlation was observed between enrichment and affinity. This work reports on the reproducibility of bacterial display in combination with FACS and provides insights into selection design to help guide the development of new affinity proteins.
authors.affiliation: Division of Protein Technology, School of Biotechnology, KTH Royal Institute of Technology, Stockholm, Sweden.
authors.forename: Mikael
authors.initials: M
authors.lastname: Åstrand
authors.affiliation: Division of Protein Technology, School of Biotechnology, KTH Royal Institute of Technology, Stockholm, Sweden Present address: Centre for Cellular and Biomolecular Research, The Donnelly Centre, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Johan
authors.initials: J
authors.lastname: Nilvebrant
authors.affiliation: Division of Protein Technology, School of Biotechnology, KTH Royal Institute of Technology, Stockholm, Sweden Present address: Department of Chemical and Biomolecular Engineering, The University of Melbourne, Parkville, Victoria, Australia.
authors.forename: Mattias
authors.initials: M
authors.lastname: Björnmalm
authors.affiliation: Division of Protein Technology, School of Biotechnology, KTH Royal Institute of Technology, Stockholm, Sweden.
authors.forename: Sarah
authors.initials: S
authors.lastname: Lindbo
authors.affiliation: Division of Protein Technology, School of Biotechnology, KTH Royal Institute of Technology, Stockholm, Sweden.
authors.forename: Sophia
authors.initials: S
authors.lastname: Hober
authors.affiliation: Division of Protein Technology, School of Biotechnology, KTH Royal Institute of Technology, Stockholm, Sweden lofblom@kth.se.
authors.forename: John
authors.initials: J
authors.lastname: Löfblom
mesh_terms: D000595:Amino Acid Sequence; D061505:Cell Surface Display Techniques / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D008958:Models, Molecular; D019151:Peptide Library; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D020893:Receptor, ErbB-3 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA; D013210:Staphylococcus / Q000235:genetics*; D020349:Surface Plasmon Resonance
chemical_list: D019151:Peptide Library; C581292:ERBB3 protein, human; D020893:Receptor, ErbB-3
keywords: ADAPT; FACS; bacterial display; directed evolution; selection strategy