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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

Nucleic acid and amino acid sequences of the olfactory receptor I7-OR from Rattus norvegicus. The 6×His tag (in bold) was added at the C-terminus of I7-OR sequence for immunological detection. The I7-OR fusion gene (about 1 Kb) was generated by PCR using genomic DNA, extracted from rat heart, and the underlined oligonucleotides.
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life-05-00770-f001: Nucleic acid and amino acid sequences of the olfactory receptor I7-OR from Rattus norvegicus. The 6×His tag (in bold) was added at the C-terminus of I7-OR sequence for immunological detection. The I7-OR fusion gene (about 1 Kb) was generated by PCR using genomic DNA, extracted from rat heart, and the underlined oligonucleotides.

Mentions: Furthermore, in order to express a receptor having a C-terminal hexahistidine (6×His) tag useful for protein immunodetection, a sequence encoding a 6×His peptide was added downstream of the I7-OR coding region in the novel pNP-I7 vector. Figure 1 shows the nucleic acid and amino acid sequences of the I7-OR from Rattus norvegicus.


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)

Nucleic acid and amino acid sequences of the olfactory receptor I7-OR from Rattus norvegicus. The 6×His tag (in bold) was added at the C-terminus of I7-OR sequence for immunological detection. The I7-OR fusion gene (about 1 Kb) was generated by PCR using genomic DNA, extracted from rat heart, and the underlined oligonucleotides.
© Copyright Policy
Related In: Results  -  Collection

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

life-05-00770-f001: Nucleic acid and amino acid sequences of the olfactory receptor I7-OR from Rattus norvegicus. The 6×His tag (in bold) was added at the C-terminus of I7-OR sequence for immunological detection. The I7-OR fusion gene (about 1 Kb) was generated by PCR using genomic DNA, extracted from rat heart, and the underlined oligonucleotides.
Mentions: Furthermore, in order to express a receptor having a C-terminal hexahistidine (6×His) tag useful for protein immunodetection, a sequence encoding a 6×His peptide was added downstream of the I7-OR coding region in the novel pNP-I7 vector. Figure 1 shows the nucleic acid and amino acid sequences of the I7-OR from Rattus norvegicus.

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