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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

Localization of reporter construct in transgenic cell cultures. Total proteins from transformed (T) and wild-type (wt) cells were precipitated with TCA and analysed by 12.5% SDS-PAGE, followed by Western blotting using anti-6×His antibodies. Fragment sizes of the molecular mass marker are indicated on the left.
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life-05-00770-f003: Localization of reporter construct in transgenic cell cultures. Total proteins from transformed (T) and wild-type (wt) cells were precipitated with TCA and analysed by 12.5% SDS-PAGE, followed by Western blotting using anti-6×His antibodies. Fragment sizes of the molecular mass marker are indicated on the left.

Mentions: To examine the presence of the I7-OR protein in Hfx. volcanii transformed cells, firstly Western blot of total cell proteins, prepared from both wild-type and transformed clones, was developed with an anti-6×His antibody (Figure 3). No immunoreactivity was detected in control sample from wild-type cells, while one immunoreactive band was observed in the case of transformed cells. It can be observed that the apparent molecular weight of the recombinant protein expressed by transformed Hfx. volcanii (about 30 kDa) is lower than the calculated molecular weight for the I7-OR protein having 6×His tag in its amino acid sequence (39 kDa).


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)

Localization of reporter construct in transgenic cell cultures. Total proteins from transformed (T) and wild-type (wt) cells were precipitated with TCA and analysed by 12.5% SDS-PAGE, followed by Western blotting using anti-6×His antibodies. Fragment sizes of the molecular mass marker are indicated on the left.
© Copyright Policy
Related In: Results  -  Collection

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

life-05-00770-f003: Localization of reporter construct in transgenic cell cultures. Total proteins from transformed (T) and wild-type (wt) cells were precipitated with TCA and analysed by 12.5% SDS-PAGE, followed by Western blotting using anti-6×His antibodies. Fragment sizes of the molecular mass marker are indicated on the left.
Mentions: To examine the presence of the I7-OR protein in Hfx. volcanii transformed cells, firstly Western blot of total cell proteins, prepared from both wild-type and transformed clones, was developed with an anti-6×His antibody (Figure 3). No immunoreactivity was detected in control sample from wild-type cells, while one immunoreactive band was observed in the case of transformed cells. It can be observed that the apparent molecular weight of the recombinant protein expressed by transformed Hfx. volcanii (about 30 kDa) is lower than the calculated molecular weight for the I7-OR protein having 6×His tag in its amino acid sequence (39 kDa).

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