title: Production, purification and characterization of recombinant, full-length human claudin-1.
abstract: The transmembrane domain proteins of the claudin superfamily are the major structural components of cellular tight junctions. One family member, claudin-1, also associates with tetraspanin CD81 as part of a receptor complex that is essential for hepatitis C virus (HCV) infection of the liver. To understand the molecular basis of claudin-1/CD81 association we previously produced and purified milligram quantities of functional, full-length CD81, which binds a soluble form of HCV E2 glycoprotein (sE2). Here we report the production, purification and characterization of claudin-1. Both yeast membrane-bound and detergent-extracted, purified claudin-1 were antigenic and recognized by specific antibodies. Analytical ultracentrifugation demonstrated that extraction with n-octyl-β-d-glucopyranoside yielded monodispersed, dimeric pools of claudin-1 while extraction with profoldin-8 or n-decylphosphocholine yielded a dynamic mixture of claudin-1 oligomers. Neither form bound sE2 in line with literature expectations, while further functional analysis was hampered by the finding that incorporation of claudin-1 into proteoliposomes rendered them intractable to study. Dynamic light scattering demonstrated that claudin-1 oligomers associate with CD81 in vitro in a defined molar ratio of 1∶2 and that complex formation was enhanced by the presence of cholesteryl hemisuccinate. Attempts to assay the complex biologically were limited by our finding that claudin-1 affects the properties of proteoliposomes. We conclude that recombinant, correctly-folded, full-length claudin-1 can be produced in yeast membranes, that it can be extracted in different oligomeric forms that do not bind sE2 and that a dynamic preparation can form a specific complex with CD81 in vitro in the absence of any other cellular components. These findings pave the way for the structural characterization of claudin-1 alone and in complex with CD81.
authors.affiliation: School of Life and Health Sciences, Aston University, Aston Triangle, Birmingham, United Kingdom.
authors.forename: Nicklas
authors.initials: N
authors.lastname: Bonander
authors.forename: Mohammed
authors.initials: M
authors.lastname: Jamshad
authors.forename: Dominik
authors.initials: D
authors.lastname: Oberthür
authors.forename: Michelle
authors.initials: M
authors.lastname: Clare
authors.forename: James
authors.initials: J
authors.lastname: Barwell
authors.forename: Ke
authors.initials: K
authors.lastname: Hu
authors.forename: Michelle J
authors.initials: MJ
authors.lastname: Farquhar
authors.forename: Zania
authors.initials: Z
authors.lastname: Stamataki
authors.forename: Helen J
authors.initials: HJ
authors.lastname: Harris
authors.forename: Karsten
authors.initials: K
authors.lastname: Dierks
authors.forename: Timothy R
authors.initials: TR
authors.lastname: Dafforn
authors.forename: Christian
authors.initials: C
authors.lastname: Betzel
authors.forename: Jane A
authors.initials: JA
authors.lastname: McKeating
authors.forename: Roslyn M
authors.initials: RM
authors.lastname: Bill
mesh_terms: D002462:Cell Membrane / Q000378:metabolism; D062445:Claudin-1 / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D057446:Hydrodynamics; D008027:Light; D008958:Models, Molecular; D011485:Protein Binding; D055550:Protein Stability; D020836:Protein Structure, Quaternary; D011510:Proteolipids / Q000378:metabolism; D011523:Protoplasts / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000378:metabolism; D012542:Scattering, Radiation; D060225:Tetraspanin 28 / Q000378:metabolism
chemical_list: D062445:Claudin-1; D011510:Proteolipids; D011994:Recombinant Proteins; D060225:Tetraspanin 28; C020485:proteoliposomes