Bacterially expressed human serotonin receptor 3A is functionally reconstituted in proteoliposomes

Abstract

Human serotonin receptor 3A (5-HT3A) is a ligand-gated ion channel regulated by serotonin. A fusion protein (P9-5-HT3A) of 5-HT3A with the P9 protein, a major envelope protein of bacteriophage phi6, was highly expressed in the membrane fraction of Escherichia coli, and the expressed protein was purified to homogeneity using an affinity chromatography. P9-5-HT3A was observed as mixed oligomers in detergents. The purified P9-5-HT3A was efficiently reconstituted into proteoliposomes, and the serotonin-dependent ion-channel activity of P9-5-HT3A was observed by measuring the increased fluorescence of Fluo-3 attributed to the formation of a complex with the Ca2+ ions released from the proteoliposomes. Alanine substitution for Trp178 of 5-HT3A abolished the serotonin-dependent ion-channel activity, confirming the importance of Trp178 as a ligand-binding site. Furthermore, the ion-channel activity of the reconstituted P9-5-HT3A was effectively blocked by treatment with ondansetron, an antagonist of 5-HT3A. The bacterial expression system of human 5-HT3A and the proteoliposomes reconstituted with 5-HT3A would provide biophysical and structural analyses of 5-HT3A.

Half a Century of Research on Membrane-Containing Bacteriophages: Bringing New Concepts to Modern Virology

“…The in vivo and in vitro dsRNA replication systems based on phi6 polymerase complex or RNA-dependent RNA polymerase enable the large-scale production of high-quality dsRNA molecules [81,128] to be used in RNA interference applications against plant and human pathogens [81,129,130,131,132,133] or to induce innate immunity responses in human cell lines [134]. In addition, the phi6 major membrane protein has been used to improve the correct folding of heterologous membrane proteins in bacterial expression systems [135,136] and to produce phi6-specific synthetic lipid scaffolds for enzymatic reactions in E. coli [137].…”

Bacteriophage membrane protein P9 as a fusion partner for the efficient expression of membrane proteins in Escherichia coli

“…In fact, we showed recently that HTR3A expressed by pMP6 is functional in the binding of its ligand [27].…”

Overexpression and Functional Stabilization of Recombinant Human Lysophosphatidic Acid Receptor 1 Using an Amphiphatic Polymer

“…Previously, we have shown that membrane proteins can be highly expressed in the membrane fraction of E . coli as fusion proteins of the P9 envelope protein of bacteriophage phi6 . An expression vector of LPA 1 was constructed as a fusion protein of P9 in which the coding sequence of LPA 1 was fused at the C‐terminus of P9, which was flanked by protease cleavage sites and His‐tag sequences at the N‐ and C‐terminus of LPA 1 , respectively (Figure (a)).…”

“…Previously, we developed an efficient expression system for membrane proteins, including GPCRs in Escherichia coli , in which a short single transmembrane envelope protein, P9, of Pseudomonas phage phi6 is fused at the N‐terminus of the target membrane protein . With this expression system, milligram quantities of GPCR, such as human endotheline receptor (ET A ) or ligand‐gated ion channel, e.g ., serotonin receptor type 5, can be easily prepared at a liter culture scale.…”