Synthesis of an oligonucleotide-derivatized amphipol and its use to trap and immobilize membrane proteins

Amphipols (APols) are specially designed amphipathic polymers that stabilize membrane proteins (MPs) in aqueous solutions in the absence of detergent. A8–35, a polyacrylate-based APol, has been grafted with an oligodeoxynucleotide (ODN). The synthesis, purification and properties of the resulting ‘OligAPol’ have been investigated. Grafting was performed by reacting an ODN carrying an amine-terminated arm with the carboxylates of A8–35. The use of OligAPol for trapping MPs and immobilizing them onto solid supports was tested using bacteriorhodopsin (BR) and the transmembrane domain of Escherichia coli outer membrane protein A (tOmpA) as model proteins. BR and OligAPol form water-soluble complexes in which BR remains in its native conformation. Hybridization of the ODN arm with a complementary ODN was not hindered by the assembly of OligAPol into particles, nor by its association with BR. BR/OligAPol and tOmpA/OligAPol complexes could be immobilized onto either magnetic beads or gold nanoparticles grafted with the complementary ODN, as shown by spectroscopic measurements, fluorescence microscopy and the binding of anti-BR and anti-tOmpA antibodies. OligAPols provide a novel, highly versatile approach to tagging MPs, without modifying them chemically nor genetically, for specific, reversible and targetable immobilization, e.g. for nanoscale applications

Labeling and Functionalizing Amphipols for Biological Applications

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Folding and stabilizing membrane proteins in amphipol A8-35

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Solubilization and stabilization of membrane proteins by cycloalkane-modified amphiphilic polymers.

International audienceMembrane proteins need to be extracted from biological membranes and purified in their native state for most structural and functional in vitro investigations. Amphiphilic copolymers, such as amphipols (APols), have emerged as very useful alternatives to detergents for keeping membrane proteins water-soluble under their native form. However, classical APols, i.e. poly(acrylic acid) (PAA) derivatives, seldom enable direct membrane protein extraction. Poly(styrene-maleic anhydride) copolymers (SMAs), which bear aromatic rings as hydrophobic side groups, have been reported to be more effective extracting agents. In order to test the hypothesis of a role of cyclic hydrophobic moieties in membrane solubilization by copolymers, we have prepared PAA derivatives comprising cyclic rather than linear aliphatic side groups (CyclAPols). As references, APol A8-35, SMAs, and diisobutylene maleic acid (DIBMA) were compared with CyclAPols. Using as models the plasma membrane of Escherichia coli and the extraction-resistant purple membrane from Halobacterium salinarum, we show that CyclAPols combine the extraction efficiency of SMAs with the stabilization afforded to membrane proteins by classical APols such as A8-35

Improved protection against Chlamydia muridarum using the native major outer membrane protein trapped in Resiquimod-carrying amphipols and effects in protection with addition of a Th1 (CpG-1826) and a Th2 (Montanide ISA 720) adjuvant.

A new vaccine formulated with the Chlamydia muridarum native major outer membrane protein (nMOMP) and amphipols was assessed in an intranasal (i.n.) challenge mouse model. nMOMP was trapped either in amphipol A8-35 (nMOMP/A8-35) or in A8-35 conjugated with Resiquimod (nMOMP/Resiq-A8-35), a TLR7/8 agonist added as adjuvant. The effects of free Resiquimod and/or additional adjuvants, Montanide ISA 720 (TLR independent) and CpG-1826 (TLR9 agonist), were also evaluated. Immunization with nMOMP/A8-35 alone administered i.n. was used as negative adjuvant-control group, whereas immunizations with C. muridarum elementary bodies (EBs) and MEM buffer, administered i.n., were used as positive and negative controls, respectively. Vaccinated mice were challenged i.n. with C. muridarum and changes in body weight, lungs weight and recovery of Chlamydia from the lungs were evaluated. All the experimental groups showed protection when compared with the negative control group. Resiquimod alone produced weak humoral and cellular immune responses, but both Montanide and CpG-1826 showed significant increases in both responses. The addition of CpG-1826 alone switched immune responses to be Th1-biased. The most robust protection was elicited in mice immunized with the three adjuvants and conjugated Resiquimod. Increased protection induced by the Resiquimod covalently linked to A8-35, in the presence of Montanide and CpG-1826 was established based on a set of parameters: (1) the ability of the antibodies to neutralize C. muridarum; (2) the increased proliferation of T-cells in vitro accompanied by higher production of IFN-γ, IL-6 and IL-17; (3) the decreased body weight loss over the 10 days after challenge; and (4) the number of IFUs recovered from the lungs at day 10 post challenge. In conclusion, a vaccine formulated with the C. muridarum nMOMP bound to amphipols conjugated with Resiquimod enhances protective immune responses that can be further improved by the addition of Montanide and CpG-1826

Influence of hydrophobic groups attached to amphipathic polymers on the solubilization of membrane proteins along with their lipids

International audienceOne of the biggest challenges in membrane protein (MP) research is to secure physiologically relevant structural and functional information after extracting MPs from their native membrane. Amphipathic polymers represent attractive alternatives to detergents for stabilizing MPs in aqueous solutions. The predominant polymers used in MP biochemistry and biophysics are amphipols (APols), one class of which, styrene maleic-acid (SMA) copolymers and their derivatives, has proven particularly efficient at MP extraction. In order to examine the relationship between the chemical structure of the polymers and their ability to extract MPs, we have developed two novel classes of APols bearing either cycloalkane or aryl (aromatic) rings, named CyclAPols and ArylAPols, respectively. The effect on solubilization of such parameters as the density of hydrophobic groups, the number of carbon atoms and their arrangement in the hydrophobic moieties, as well as the charge density of the polymers was evaluated. The membrane-solubilizing efficiency of the SMAs, CyclAPols and ArylAPols was compared using as models i) two MPs, BmrA and a GFP-fused version of LacY, overexpressed in the inner membrane of Escherichia coli, and ii) bacteriorhodopsin, naturally expressed in the purple membrane of Halobacterium salinarum. This analysis shows that, as compared to SMAs, the novel APols feature an improved efficiency at extracting MPs while preserving native protein-lipid interactions

Improved protection against Chlamydia muridarum using the native major outer membrane protein trapped in Resiquimod-carrying amphipols and effects in protection with addition of a Th1 (CpG-1826) and a Th2 (Montanide ISA 720) adjuvant

A new vaccine formulated with the Chlamydia muridarum native major outer membrane protein (nMOMP) and amphipols was assessed in an intranasal (i.n.) challenge mouse model. nMOMP was trapped either in amphipol A8-35 (nMOMP/A8-35) or in A8-35 conjugated with Resiquimod (nMOMP/Resiq-A8-35), a TLR7/8 agonist added as adjuvant. The effects of free Resiquimod and/or additional adjuvants, Montanide ISA 720 (TLR independent) and CpG-1826 (TLR9 agonist), were also evaluated. Immunization with nMOMP/A8-35 alone administered i.n. was used as negative adjuvant-control group, whereas immunizations with C. muridarum elementary bodies (EBs) and MEM buffer, administered i.n., were used as positive and negative controls, respectively. Vaccinated mice were challenged i.n. with C. muridarum and changes in body weight, lungs weight and recovery of Chlamydia from the lungs were evaluated. All the experimental groups showed protection when compared with the negative control group. Resiquimod alone produced weak humoral and cellular immune responses, but both Montanide and CpG-1826 showed significant increases in both responses. The addition of CpG-1826 alone switched immune responses to be Th1-biased. The most robust protection was elicited in mice immunized with the three adjuvants and conjugated Resiquimod. Increased protection induced by the Resiquimod covalently linked to A8-35, in the presence of Montanide and CpG-1826 was established based on a set of parameters: (1) the ability of the antibodies to neutralize C. muridarum; (2) the increased proliferation of T-cells in vitro accompanied by higher production of IFN-γ, IL-6 and IL-17; (3) the decreased body weight loss over the 10 days after challenge; and (4) the number of IFUs recovered from the lungs at day 10 post challenge. In conclusion, a vaccine formulated with the C. muridarum nMOMP bound to amphipols conjugated with Resiquimod enhances protective immune responses that can be further improved by the addition of Montanide and CpG-1826

Co-delivery of amphipol-conjugated adjuvant with antigen, and adjuvant combinations, enhance immune protection elicited by a membrane protein-based vaccine against a mucosal challenge with Chlamydia

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