title: High-throughput engineering and analysis of peptide binding to class II MHC.
abstract: Class II major histocompatibility complex (MHC-II) proteins govern stimulation of adaptive immunity by presenting antigenic peptides to CD4+ T lymphocytes. Many allelic variants of MHC-II exist with implications in peptide presentation and immunity; thus, high-throughput experimental tools for rapid and quantitative analysis of peptide binding to MHC-II are needed. Here, we present an expression system wherein peptide and MHC-II are codisplayed on the surface of yeast in an intracellular association-dependent manner and assayed by flow cytometry. Accordingly, the relative binding of different peptides and/or MHC-II variants can be assayed by genetically manipulating either partner, enabling the application of directed evolution approaches for high-throughput characterization or engineering. We demonstrate the application of this tool to map the side-chain preference for peptides binding to HLA-DR1 and to evolve novel HLA-DR1 mutants with altered peptide-binding specificity.
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia, PA 19104, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Jiang
authors.forename: Eric T
authors.initials: ET
authors.lastname: Boder
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D019020:Directed Molecular Evolution; D005434:Flow Cytometry; D015798:HLA-DR1 Antigen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D015003:Yeasts / Q000235:genetics / Q000378:metabolism
chemical_list: D015798:HLA-DR1 Antigen; D019151:Peptide Library; D010455:Peptides