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Production, Purific...
Production, Purification and Characterization of Recombinant, Full-Length Human Claudin-1
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- Bonander, Nicklas, 1968 (author)
- Chalmers tekniska högskola,Chalmers University of Technology
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- Jamshad, M. (author)
- Aston University
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- Oberthür, D. (author)
- Center for Free-Electron Laser Science (CFEL),Universität Hamburg,University of Hamburg
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- Clare, M. (author)
- Aston University
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- Barwell, J. (author)
- Aston University
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- Hu, K. (author)
- University of Birmingham
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- Farquhar, M.J. (author)
- University of Birmingham
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- Stamataki, Z. (author)
- University of Birmingham
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- Harris, H.J. (author)
- University of Birmingham
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- Dierks, K. (author)
- Universität Hamburg,University of Hamburg
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- Dafforn, T.R. (author)
- University of Birmingham
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- Betzel, C. (author)
- Universität Hamburg,University of Hamburg
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- McKeating, J.A. (author)
- University of Birmingham
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- Bill, R (author)
- Aston University
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(creator_code:org_t)
- 2013-05-21
- 2013
- English.
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In: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203 .- 1932-6203. ; 8:5, s. Art. no. e64517-
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Abstract
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- 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.
Subject headings
- NATURVETENSKAP -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)
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- art (subject category)
- ref (subject category)
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- By the author/editor
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Bonander, Nickla ...
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Jamshad, M.
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Oberthür, D.
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Clare, M.
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Barwell, J.
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Hu, K.
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show more...
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Farquhar, M.J.
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Stamataki, Z.
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Harris, H.J.
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Dierks, K.
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Dafforn, T.R.
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Betzel, C.
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McKeating, J.A.
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Bill, R
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- NATURAL SCIENCES
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NATURAL SCIENCES
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and Biological Scien ...
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and Biochemistry and ...
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PLoS ONE
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Chalmers University of Technology