A renewed global commitment to malaria elimination lends urgency to understanding the biology of Plasmodium transmission stages. Recent progress toward uncovering the mechanisms underlying Plasmodium falciparum sexual differentiation and maturation reveals potential targets for transmission-blocking drugs and vaccines. The identification of parasite factors that alter sexual differentiation, including extracellular vesicles and a master transcriptional regulator, suggest that parasites make epigenetically controlled developmental decisions based on environmental cues. New insights into sexual development, especially host cell remodeling and sequestration in the bone marrow, highlight open questions regarding parasite homing to the tissue, transmigration across the vascular endothelium, and maturation in the parenchyma. Novel molecular and translational tools will provide further opportunities to define host-parasite interactions and design effective transmission-blocking therapeutics.
Host-Pathogen Interactions , Plasmodium falciparum/physiology , Animals , Epigenesis, Genetic , Gene Expression Regulation , Humans , Plasmodium falciparum/growth & development
A crucial bottleneck in membrane protein studies, particularly G-protein coupled receptors, is the notorious difficulty of finding an optimal detergent that can solubilize them and maintain their stability and function. Here we report rapid production of 12 unique mammalian olfactory receptors using short designer lipid-like peptides as detergents. The peptides were able to solubilize and stabilize each receptor. Circular dichroism showed that the purified olfactory receptors had alpha-helical secondary structures. Microscale thermophoresis suggested that the receptors were functional and bound their odorants. Blot intensity measurements indicated that milligram quantities of each olfactory receptor could be produced with at least one peptide detergent. The peptide detergents' capability was comparable to that of the detergent Brij-35. The ability of 10 peptide detergents to functionally solubilize 12 olfactory receptors demonstrates their usefulness as a new class of detergents for olfactory receptors, and possibly other G-protein coupled receptors and membrane proteins.
Detergents/chemistry , Lipids/chemistry , Peptides/chemistry , Receptors, Odorant/metabolism , Animals , Cell-Free System , Circular Dichroism , Humans , Hydrogen-Ion Concentration , Ligands , Mice , Models, Molecular , Polyethylene Glycols/chemistry , Protein Structure, Secondary , Receptors, Odorant/chemistry , Receptors, Odorant/isolation & purification , Silver Staining , Solubility , Temperature
Membrane proteins, particularly G-protein coupled receptors (GPCRs), are notoriously difficult to express. Using commercial E. coli cell-free systems with the detergent Brij-35, we could rapidly produce milligram quantities of 13 unique GPCRs. Immunoaffinity purification yielded receptors at >90% purity. Secondary structure analysis using circular dichroism indicated that the purified receptors were properly folded. Microscale thermophoresis, a novel label-free and surface-free detection technique that uses thermal gradients, showed that these receptors bound their ligands. The secondary structure and ligand-binding results from cell-free produced proteins were comparable to those expressed and purified from HEK293 cells. Our study demonstrates that cell-free protein production using commercially available kits and optimal detergents is a robust technology that can be used to produce sufficient GPCRs for biochemical, structural, and functional analyses. This robust and simple method may further stimulate others to study the structure and function of membrane proteins.
Receptors, G-Protein-Coupled/metabolism , Cell Line , Electrophoresis, Polyacrylamide Gel , Humans , Ligands , Protein Conformation , Protein Structure, Secondary , Receptors, G-Protein-Coupled/chemistry , Receptors, G-Protein-Coupled/physiology , Solubility
