Selective detergent-extraction from mixed detergent/lipid/protein micelles, using cyclodextrin inclusion compounds: a novel generic approach for the preparation of proteoliposomes

Abstract

A novel generic approach is described for the selective extraction of detergents from mixed detergent/lipid/protein micelles for the preparation of proteoliposomes of defined lipid-protein ratio. The approach is based on the much higher affinity of inclusion compounds of the cyclodextrin type for detergents in comparison with bilayer-forming lipids. This approach has distinct advantages over other procedures currently in use. It produces good results with all detergents tested, independent of type and critical micelle concentration, and appears to be generally applicable. It yields nearly quantitative recovery of membrane protein in the proteoliposome fraction. Finally, no large excess of lipid is required; a molar ratio of lipid to protein of 100 to 1 already produces proteoliposomes with functional membrane protein, but higher ratios are well tolerated. The size of the vesicles thus obtained depends on the detergent used. Separation of the resulting proteoliposomes from the detergent-cyclodextrin complexes was most easily achieved by centrifugation through a discontinuous sucrose gradient. A variety of detergents was tested in this procedure on the bovine rod visual pigment rhodopsin in combination with retina lipids. In all cases good yields of proteoliposomes were obtained, which contained fully functional rhodopsin.

Recombinant Cannabinoid Type 2 Receptor in Liposome Model Activates G Protein in Response to Anionic Lipid Constituents

“…Materials-The lipids POPC, POPC-d 31 , POPC-d 4 , POPS sodium salt, and cholesterol were purchased from Avanti Polar FIGURE 1. Snake plot of the human CB 2 receptor.…”

Molecular Characterization of Vibrio parahaemolyticusvSGLT

“…Freeze-Fracture Electron Microscopy of Liposome-reconstituted VNH6A-Purified VNH6A was reconstituted into PLs (31). Aliquots (ϳ5 l) of PLs, as prepared for uptake assays, were treated for freeze-fracture EM.…”

GRecon: A Method for the Lipid Reconstitution of Membrane Proteins

“…More recently, cyclodextrins, cyclic sugar oligomers of 950-1300 Da, have been used to remove detergents from membrane protein solutions. [6,7] Cyclodextrins work well with most commonly used detergents, but have the same disadvantage as Bio-Beads in that detergent removal can be too rapid and difficult to control. A problem common to all these methods is that they produce both proteoliposomes and empty liposomes, which ideally need to be removed.…”

“…This may be desirable in special cases, but the high affinity of b-cyclodextrin precludes the use of Triton X-100 for destabilizing preformed liposomes. [6] In summary, GRecon offers a number of decisive advantages over conventional reconstitution procedures: 1) As a single-step method it is quick and convenient, requiring minimal manual sample handling. 2) Reconstitution works reliably and results are reproducible.…”

“…In most cases a final molar ratio of 1.2:1 to 2.4:1 (cyclodextrin/detergent) is sufficient. [6] Detergent removal starts slowly and then progresses as the protein migrates into the gradient. By contrast, the rate of detergent removal by dialysis or absorption in bulk solution is highest at the outset, when the concentration of detergent monomers is at its maximum, and then decreases.…”