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

Lobasso S, Vitale R, Lopalco P, Corcelli A - Life (Basel) (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Basic Medical Sciences, Neurosciences and Sensory Organs, University of Bari "Aldo Moro", Piazza Giulio Cesare 11, I-70124 Bari, Italy. simona.lobasso@uniba.it.

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.

No MeSH data available.


Related in: MedlinePlus

mRNA of the rat I7-OR in transformed Hfx. volcanii strain. Total RNA was extracted from Hfx. volcanii cells transformed with pNP-I7 expression vector and subjected to RT-PCR using specific primers. (1) PCR blank; (2) PCR negative control (absence of RT enzyme in the mixture); (3) cDNA of Hfx. volcanii; (M) molecular mass marker.
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life-05-00770-f002: mRNA of the rat I7-OR in transformed Hfx. volcanii strain. Total RNA was extracted from Hfx. volcanii cells transformed with pNP-I7 expression vector and subjected to RT-PCR using specific primers. (1) PCR blank; (2) PCR negative control (absence of RT enzyme in the mixture); (3) cDNA of Hfx. volcanii; (M) molecular mass marker.

Mentions: Figure 2 shows the RT-PCR analysis conducted on RNA extracted from the transformed strain: it can be seen that RT-PCR product for the I7-OR was found at expected size (about 1000 bp). No such transcript was detected in a negative control.


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

Lobasso S, Vitale R, Lopalco P, Corcelli A - Life (Basel) (2015)

mRNA of the rat I7-OR in transformed Hfx. volcanii strain. Total RNA was extracted from Hfx. volcanii cells transformed with pNP-I7 expression vector and subjected to RT-PCR using specific primers. (1) PCR blank; (2) PCR negative control (absence of RT enzyme in the mixture); (3) cDNA of Hfx. volcanii; (M) molecular mass marker.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4390878&req=5

life-05-00770-f002: mRNA of the rat I7-OR in transformed Hfx. volcanii strain. Total RNA was extracted from Hfx. volcanii cells transformed with pNP-I7 expression vector and subjected to RT-PCR using specific primers. (1) PCR blank; (2) PCR negative control (absence of RT enzyme in the mixture); (3) cDNA of Hfx. volcanii; (M) molecular mass marker.
Mentions: Figure 2 shows the RT-PCR analysis conducted on RNA extracted from the transformed strain: it can be seen that RT-PCR product for the I7-OR was found at expected size (about 1000 bp). No such transcript was detected in a negative control.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Basic Medical Sciences, Neurosciences and Sensory Organs, University of Bari "Aldo Moro", Piazza Giulio Cesare 11, I-70124 Bari, Italy. simona.lobasso@uniba.it.

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.

No MeSH data available.


Related in: MedlinePlus