Tetraspanins are integral membrane proteins that play a key role in organising multi-molecular complexes. Tetraspanin CD81 is involved in cellular processes such as cell adhesion, cell proliferation, and mediating infection of medically important pathogens, including the hepatitis C virus. Despite these important roles, the comprehensive structural organisation, membrane distribution, and CD81 interaction with surrounding proteins and lipids are not known. To enable these studies, CD81 was solubilised and purified from Pichia pastoris membranes in poly (styrene-co-maleic-acid) lipid particles or SMALPs, to retain its surrounding membrane environment. Biophysical characterisation was conducted by circular dichroism spectroscopy and antigen-antibody ELISA. This indicated that SMALP-CD81 retains its secondary structure and is functionally stable, even at higher temperatures, in marked contrast with detergent-purified CD81. Subsequently, gel filtration conditions have been optimised to isolate functionally active SMALP-CD81 fractions. The native CD81 membrane distribution in HEK 293 and Huh-7 cell-lines was also studied using electron microscopy (EM). The EM images of cell sections indicated that CD81 is organised in isolated monomers as well as in clusters of potentially higher-order structures in both cell-lines. This data agrees with the general consensus that tetraspanins exist as tetraspanin-rich microdomains that modulate the function of interacting proteins.
- immobilized metal affinity chromatography
- size exclusion chromatography
- recombinant protein production
- lipidic cubic phase
- mammalian cells