title: Direct expression and validation of phage-selected peptide variants in mammalian cells.
abstract: Phage display is a key technology for the identification and maturation of high affinity peptides, antibodies, and other proteins. However, limitations of bacterial expression restrict the range and sensitivity of assays that can be used to evaluate phage-selected variants. To address this problem, selected genes are typically transferred to mammalian expression vectors, a major rate-limiting step in the iterative improvement of peptides and proteins. Here we describe a system that combines phage display and efficient mammalian expression in a single vector, pDQ1. This system permits immediate expression of phage-selected genes as IgG1-Fc fusions in mammalian cells, facilitating the rapid, sensitive characterization of a large number of library outputs for their biochemical and functional properties. We demonstrate the utility of this system by improving the ability of a CD4-mimetic peptide to bind the HIV-1 envelope glycoprotein and neutralize HIV-1 entry. We further improved the potency of the resulting peptide, CD4mim6, by limiting its ability to induce the CD4-bound conformation of the envelope glycoprotein. Thus, CD4mim6 and its variants can be used to investigate the properties of the HIV-1 envelope glycoprotein, and pDQ1 can accelerate the discovery of new peptides and proteins through phage display.
authors.affiliation: Department of Infectious Diseases, The Scripps Research Institute, Jupiter, Florida 33458, USA. bquinlan@scripps.edu
authors.forename: Brian D
authors.initials: BD
authors.lastname: Quinlan
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Gardner
authors.forename: Vinita R
authors.initials: VR
authors.lastname: Joshi
authors.forename: Jessica J
authors.initials: JJ
authors.lastname: Chiang
authors.forename: Michael
authors.initials: M
authors.lastname: Farzan
mesh_terms: D000595:Amino Acid Sequence; D000906:Antibodies / Q000737:chemistry; D000998:Antiviral Agents / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods; D005822:Genetic Vectors; D057809:HEK293 Cells; D015497:HIV-1 / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019151:Peptide Library; D019713:Receptors, CCR5 / Q000378:metabolism; D020349:Surface Plasmon Resonance; D053586:Virus Internalization / Q000187:drug effects; D054299:env Gene Products, Human Immunodeficiency Virus / Q000037:antagonists & inhibitors*
chemical_list: D000906:Antibodies; D000998:Antiviral Agents; D019151:Peptide Library; D019713:Receptors, CCR5; D054299:env Gene Products, Human Immunodeficiency Virus
keywords: Antimicrobial Peptides; Antiviral Agents; Fc Fusion Protein; HIV-1; Human Immunodeficiency Virus; Immunoadhesin; Phage Display