Production, purification and characterization of recombinant, full-length human claudin-1

Nicklas Bonander, Mohammed Jamshad, Dominik Oberthür, Michelle Clare, James Barwell, Ke Hu, Michelle J. Farquhar, Zania Stamataki, Helen J. Harris, Karsten Dierks, Timothy R. Dafforn, Christian Betzel, Jane A. McKeating, Roslyn M. Bill

Research output: Contribution to journalArticle

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-ß-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.
Original languageEnglish
Article numbere64517
Number of pages12
JournalPLoS ONE
Volume8
Issue number5
DOIs
Publication statusPublished - 21 May 2013

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Claudin-1
Purification
Hepatitis C virus
yeasts
tight junctions
ultracentrifugation
light scattering
detergents
glycoproteins
liver
receptors
antibodies
assays
Oligomers
Yeast
infection
Yeasts
Claudins
proteins
Membranes

Bibliographical note

© 2013 Bonander et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: European Commission (contracts LSHG-CT-2006-037793 (OptiCryst) and LSHG-CT-2004-504601 (E-MeP)); Wellcome Trust (Grant 14101 and ISSF award); and BBSRC (studentship); Royal Society (fellowship); Röntgen-Angström-Cluster (Project 05K12GU3) and the German Federal Ministry of Education and Research (BMBF).

Keywords

  • CD81 antigens
  • cell membrane
  • claudin-1
  • humans
  • hydrodynamics
  • light
  • molecular models
  • protein binding
  • protein stability
  • quaternary protein structure
  • proteolipids
  • protoplasts
  • recombinant proteins
  • saccharomyces cerevisiae
  • radiation scattering

Cite this

Bonander, Nicklas ; Jamshad, Mohammed ; Oberthür, Dominik ; Clare, Michelle ; Barwell, James ; Hu, Ke ; Farquhar, Michelle J. ; Stamataki, Zania ; Harris, Helen J. ; Dierks, Karsten ; Dafforn, Timothy R. ; Betzel, Christian ; McKeating, Jane A. ; Bill, Roslyn M. / Production, purification and characterization of recombinant, full-length human claudin-1. In: PLoS ONE. 2013 ; Vol. 8, No. 5.
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Bonander, N, Jamshad, M, Oberthür, D, Clare, M, Barwell, J, Hu, K, Farquhar, MJ, Stamataki, Z, Harris, HJ, Dierks, K, Dafforn, TR, Betzel, C, McKeating, JA & Bill, RM 2013, 'Production, purification and characterization of recombinant, full-length human claudin-1', PLoS ONE, vol. 8, no. 5, e64517. https://doi.org/10.1371/journal.pone.0064517

Production, purification and characterization of recombinant, full-length human claudin-1. / Bonander, Nicklas; Jamshad, Mohammed; Oberthür, Dominik; Clare, Michelle; Barwell, James; Hu, Ke; Farquhar, Michelle J.; Stamataki, Zania; Harris, Helen J.; Dierks, Karsten; Dafforn, Timothy R.; Betzel, Christian; McKeating, Jane A.; Bill, Roslyn M.

In: PLoS ONE, Vol. 8, No. 5, e64517, 21.05.2013.

Research output: Contribution to journalArticle

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AU - Bonander, Nicklas

AU - Jamshad, Mohammed

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AU - Clare, Michelle

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AU - Hu, Ke

AU - Farquhar, Michelle J.

AU - Stamataki, Zania

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AU - Dafforn, Timothy R.

AU - Betzel, Christian

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AU - Bill, Roslyn M.

N1 - © 2013 Bonander et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: European Commission (contracts LSHG-CT-2006-037793 (OptiCryst) and LSHG-CT-2004-504601 (E-MeP)); Wellcome Trust (Grant 14101 and ISSF award); and BBSRC (studentship); Royal Society (fellowship); Röntgen-Angström-Cluster (Project 05K12GU3) and the German Federal Ministry of Education and Research (BMBF).

PY - 2013/5/21

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N2 - 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-ß-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.

AB - 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-ß-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.

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Bonander N, Jamshad M, Oberthür D, Clare M, Barwell J, Hu K et al. Production, purification and characterization of recombinant, full-length human claudin-1. PLoS ONE. 2013 May 21;8(5). e64517. https://doi.org/10.1371/journal.pone.0064517