Production, Purification and Characterization of Recombinant, Full-Length Human Claudin-1
Artikel i vetenskaplig tidskrift, 2013

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.

Författare

Nicklas Bonander

Chalmers, Kemi- och bioteknik, Industriell Bioteknik

M. Jamshad

Aston University

D. Oberthür

Universitat Hamburg

Center for Free-Electron Laser Science

M. Clare

Aston University

J. Barwell

Aston University

K. Hu

University of Birmingham

M.J. Farquhar

University of Birmingham

Z. Stamataki

University of Birmingham

H.J. Harris

University of Birmingham

K. Dierks

Universitat Hamburg

T.R. Dafforn

University of Birmingham

C. Betzel

Universitat Hamburg

J.A. McKeating

University of Birmingham

R Bill

Aston University

PLoS ONE

1932-6203 (ISSN)

Vol. 8 Art. no. e64517- e64517

Ämneskategorier

Biokemi och molekylärbiologi

DOI

10.1371/journal.pone.0064517