title: A novel screening system for claudin binder using baculoviral display.
abstract: Recent progress in cell biology has provided new insight into the claudin (CL) family of integral membrane proteins, which contains more than 20 members, as a target for pharmaceutical therapy. Few ligands for CL have been identified because it is difficult to prepare CL in an intact form. In the present study, we developed a method to screen for CL binders by using the budded baculovirus (BV) display system. CL4-displaying BV interacted with a CL4 binder, the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE), but it did not interact with C-CPE that was mutated in its CL4-binding region. C-CPE did not interact with BV and CL1-displaying BV. We used CL4-displaying BV to select CL4-binding phage in a mixture of a scFv-phage and C-CPE-phage. The percentage of C-CPE-phage in the phage mixture increased from 16.7% before selection to 92% after selection, indicating that CL-displaying BV may be useful for the selection of CL binders. We prepared a C-CPE phage library by mutating the functional amino acids. We screened the library for CL4 binders by affinity to CL4-displaying BV, and we found that the novel CL4 binders modulated the tight-junction barrier. These findings indicate that the CL-displaying BV system may be a promising method to produce a novel CL binder and modulator.
authors.affiliation: Laboratory of Bio-Functional Molecular Chemistry, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka, Japan.
authors.forename: Hideki
authors.initials: H
authors.lastname: Kakutani
authors.forename: Azusa
authors.initials: A
authors.lastname: Takahashi
authors.forename: Masuo
authors.initials: M
authors.lastname: Kondoh
authors.forename: Yumiko
authors.initials: Y
authors.lastname: Saito
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Yamaura
authors.forename: Toshiko
authors.initials: T
authors.lastname: Sakihama
authors.forename: Takao
authors.initials: T
authors.lastname: Hamakubo
authors.forename: Kiyohito
authors.initials: K
authors.lastname: Yagi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D018938:Caco-2 Cells; D002478:Cells, Cultured; D062506:Claudin-4; D004768:Enterotoxins / Q000737:chemistry / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D019151:Peptide Library; D011485:Protein Binding / Q000502:physiology; D025941:Protein Interaction Mapping / Q000379:methods; D018411:Spodoptera
chemical_list: C568157:CLDN4 protein, human; D062506:Claudin-4; D004768:Enterotoxins; D008024:Ligands; D008565:Membrane Proteins; D010446:Peptide Fragments; D019151:Peptide Library; C035598:enterotoxin, Clostridium