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.

Functional recombinant protein is present in the pre-induction phases of Pichia pastoris cultures when grown in bioreactors, but not shake-flasks

“…In contrast, shake-flask cultivations of GFP [22], HRP, hCD81 [23], hCD82 [23] and human claudin-1 [23] showed no recombinant protein production in the pre-induction phases (Table 2, Additional file 1). …”

CD81 extracted in SMALP nanodiscs comprises two distinct protein populations within a lipid environment enriched with negatively charged headgroups

“…Presumably the void peak contains some form of larger assembly or aggregated species, however it is notable that both peaks contain fully folded CD81, thus the void peak does not contain denatured, aggregated protein. CD81 itself is known to form dimers and larger assemblies [ 4 , 5 ] so it is possible that this peak contains larger assemblies of CD81. Notably, the CD81 crystal structure revealed a monomeric form of a tetraspanin, which contrasts with the crystal structure of the extracellular LEL domain of human CD81 (88 of 236 residues).…”

“…The family's signature motif is the presence of four or more cysteine residues in LEL; two in highly-conserved ‘CCG’ motifs [ 1 ]. Central to tetraspanin function are their extensive associations with an array of signalling proteins [ 1 ]; tetraspanin homodimers have been proposed to be the building blocks for the assembly of multicomponent tetraspanin protein complexes [ 4 , 5 ]. The 6 Å cryo-electron microscopy structure of a uroplakin tetraspanin revealed a rod-shaped structural morphology consisting of four TM helical bundles bound to a single TM helix partner [ 6 ].…”

Computational Modeling of Hepatitis C Virus Envelope Glycoprotein Structure and Recognition

“…Related scavenger receptor CD36 also has a crystal structure available (PDB code: 5LGD) ( 110 ), and was recently proposed as an additional coreceptor that binds E1 ( 111 ). Several studies have examined the structural determinants of the CLDN1–CD81 interface ( 97 , 112 , 113 ). In silico mutagenesis of this interface revealed key binding residues ( 67 ), and MD simulations of CLDN1 point mutations showed disruptions of receptor structure thought to diminish HCV entry ( 114 ).…”