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Production, Purification and Characterization of Recombinant, Full-Length Human Claudin-1

Nicklas Bonander (Institutionen för kemi- och bioteknik, Industriell Bioteknik ) ; M. Jamshad ; D. Oberthür ; M. Clare ; J. Barwell ; K. Hu ; M.J. Farquhar ; Z. Stamataki ; H.J. Harris ; K. Dierks ; T.R. Dafforn ; C. Betzel ; J.A. McKeating ; R.M. Bill
PLoS ONE (1932-6203). Vol. 8 (2013), 5, p. Art. no. e64517.
[Artikel, refereegranskad vetenskaplig]

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.



Denna post skapades 2013-05-29. Senast ändrad 2016-07-15.
CPL Pubid: 177610

 

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Institutioner (Chalmers)

Institutionen för kemi- och bioteknik, Industriell Bioteknik (2008-2014)

Ämnesområden

Biokemi och molekylärbiologi

Chalmers infrastruktur