title: Efficient production of antibodies against a mammalian integral membrane protein by phage display.
abstract: The application of phage display technology to mammalian proteins with multiple transmembrane regions has had limited success due to the difficulty in generating these proteins in sufficient amounts and purity. We report here a method that can be easily and generally applied to sorting of phage display libraries with multispan protein targets solubilized in detergent. A key feature of this approach is the production of biotinylated multispan proteins in virions of a baculovirus vector that allows library panning without prior purification of the target protein. We obtained Fab fragments from a naïve synthetic antibody phage library that, when engineered into full-length immunoglobulin (Ig)G, specifically bind cells expressing claudin-1, a protein with four transmembrane regions that is used as an entry co-receptor by the hepatitis C virus (HCV). Affinity-matured variants of one of these antibodies efficiently inhibited HCV infection. The use of baculovirus particles as a source of mammalian multispan protein facilitates the application of phage display to this difficult class of proteins.
authors.affiliation: Departments of Antibody Engineering, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA. ihotzel@gene.com
authors.forename: Isidro
authors.initials: I
authors.lastname: Hötzel
authors.forename: Vicki
authors.initials: V
authors.lastname: Chiang
authors.forename: Jingyu
authors.initials: J
authors.lastname: Diao
authors.forename: Homer
authors.initials: H
authors.lastname: Pantua
authors.forename: Henry R
authors.initials: HR
authors.lastname: Maun
authors.forename: Sharookh B
authors.initials: SB
authors.lastname: Kapadia
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D062445:Claudin-1; D005434:Flow Cytometry; D057809:HEK293 Cells; D016174:Hepacivirus; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000235:genetics; D007074:Immunoglobulin G / Q000096:biosynthesis* / Q000235:genetics; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D019151:Peptide Library; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D016415:Sequence Alignment; D019809:Streptavidin; D014771:Virion / Q000737:chemistry / Q000378:metabolism
chemical_list: C568108:CLDN1 protein, human; D062445:Claudin-1; C568113:Cldn1 protein, mouse; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D008565:Membrane Proteins; D019151:Peptide Library; D019809:Streptavidin