Sequential purification and characterization of Torpedo californica nAChR-DC supplemented with CHS for high-resolution crystallization studies

Drosophila nicotinic acetylcholine receptor subunits and their native interactions with insecticidal peptide toxins

“…Resistance to spinosad is strongly associated with Dα6 (Perry et al, 2021), and spinosad binding is much more sensitive to the action of α-Btx than to the action of neonicotinoids (Chambers et al, 2019), again consistent with the involvement of this subunit with sensitivity to injected α-Btx and with the proposition that α-Btx and Hv1a act at distinct receptor classes. nAChR subunits are known to be difficult to purify due to solubilisation issues (Cheng et al, 2015;Maldonado-Hernández et al, 2020) and the requirement for a lipid environment for ligand binding (Dacosta et al, 2013) makes it challenging to study these receptors in native conditions. We used the SMALPs extraction method for preparing membrane discs and enriched nAChRs via α-Btx affinity purification.…”

Pursuing High-Resolution Structures of Nicotinic Acetylcholine Receptors: Lessons Learned from Five Decades

“…Recently, our laboratory developed a novel, straightforward, and sequential automated purification process to obtain highly pure and functional nAChR-DCs [ 77 , 78 ]. First, the protein is solubilized using LysoFos Choline 16 (LFC-16), a phospholipid analog detergent, supplemented with CHS, and sodium chloride at high concentrations to assist the solubilization process, thus removing impurities that have weak or non-specific interactions with the nAChR.…”

“…Moreover, the column was placed within an insulated jacket using a recirculation system to control the temperature inside the column, in addition to performing experiments in a cold room. The combination of all of these steps and strategies has substantially improved the quality and functional parameters of purified nAChR-DCs as a possible final product for the production of high-quality crystals [ 77 , 78 ]. It has been postulated that the main impediment to achieving a high-resolution structure of the nAChR is the purity, homogeneity, function, and stability of the nAChR-DCs [ 52 , 54 , 55 , 73 , 79 , 80 , 81 , 82 , 83 ].…”

“…However, few have focused on the importance of generating functional nAChR detergent complexes. The above-described purification protocols proved capable of producing a stable and functional nAChR-DC, suitable as a starting material for crystallization studies [ 77 , 78 ]. Among the most common methods used in these cases are sucrose density gradient, alkali treatment, ion exchange, gel filtration, and different affinity chromatography methodologies using nAChR ligands [ 62 , 84 , 85 , 86 ], some of which have the disadvantage of short lifespans one (1) month or less [ 87 ].…”

“…Another organism used to obtain nAChR was Electrophorus electricus (Ee), from which only 3–4 mg of purified nAChR was recovered from 500 g of tissue [ 93 ]. In our method, a significant reduction in the amount of Tc tissue used for solubilization and purification (20–40 g) is possible, from which we were able to produce 2–4 mg of pure, stable, and functional nAChRs [ 77 , 78 ]. As mentioned above, procedures that have been applied to the purification of nAChR from other tissue sources have invariably yielded nAChRs with several co-purifying contaminants.…”