Haloferax volcanii, as a Novel Tool for Producing Mammalian Olfactory Receptors Embedded in Archaeal Lipid Bilayer.

Simona Lobasso,Rita Vitale,Angela Corcelli,Patrizia Lopalco

doi:10.3390/life5010770

Abstract

The aim of this study was to explore the possibility of using an archaeal microorganism as a host system for expressing mammalian olfactory receptors (ORs). We have selected the archaeon Haloferax volcanii as a cell host system and one of the most extensively investigated OR, namely I7-OR, whose preferred ligands are short-chain aldehydes, such as octanal, heptanal, nonanal. A novel plasmid has been constructed to express the rat I7-OR, fused with a hexahistidine-tag for protein immunodetection. The presence of the recombinant receptor at a membrane level was demonstrated by immunoblot of the membranes isolated from the transgenic archaeal strain. In addition, the lipid composition of archaeonanosomes containing ORs has been characterized in detail by High-Performance Thin-Layer Chromatography (HPTLC) in combination with Matrix-Assisted Laser Desorption Ionization—Time-Of-Flight/Mass Spectrometry (MALDI-TOF/MS) analysis.

Highlights

Olfactory receptors (ORs) are members of the large super family of G-protein coupled receptors (GPCRs) [1,2,3]
In order to express ORs in the membranes of haloarchaea, we adapted strategies that had been previously developed for heterologous expression of other GPCRs in Halobacterium salinarum and Haloferax volcanii [15,16]
The plasmid utilized the strong aloarchaeal synthetic promoter PrR16 and contained a haloarchaeal replication origin; the selection of the plasmid was based on the presence of the mutated GyrB sequence, which allowed growth of transformed microorganisms on media containing the antibiotic novobiocin [16]

Summary

Introduction

Olfactory receptors (ORs) are members of the large super family of G-protein coupled receptors (GPCRs) [1,2,3]. ORs’ main biological function is the molecular recognition of odorant molecules that can disperse into the air. We have used the rat I7-OR as a model to investigate ORs expression in archaeal microorganisms, as it is one of the most extensively investigated OR. It has been cloned, expressed in neurons, functionally characterized as activated by short-chain aldehydes, such as octanal, heptanal, and nonanal [6,7], and studied by computational modeling [8,9]

Objectives

Methods

Results