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Olfactory receptor proteins. Expression, characterization and partial purification
1Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot, Israel.
Abstract:
A rat olfactory epithelium cDNA library was screened for olfactory receptor clones. One of the positively hybridizing cDNA clones was sequenced and found to encode a new member of the olfactory receptor superfamily. This cDNA, termed olp4, was used as a model of olfactory receptor for expression, both in vitro and in vivo. Expression of olp4, as well as of another previously cloned olfactory receptor (F5), was monitored by immunoprecipitation was a monoclonal antibody directed against a Flag peptide epitope tag, inserted at the N-terminus of the open reading frame, and a specific polyclonal antibody against a C-terminal peptide of olp4. Translation in vitro, followed by immunoprecipitation, showed a major olp4-specific band of 27-29 kDa. The olp4 and F5 polypeptides were found to be inserted into microsomal membranes as expected for integral membrane proteins. Expression in vivo of Flag-olp4 in Sf9 insect cells, using the baculovirus expression system, showed a specific polypeptide of the same size as the in vitro species, with an additional band of 34 kDa, which is most likely a glycosylated form. Fluorescence cytometry and immunohistochemical assays demonstrated the localization of the Flag-olp4 product on the cell surface of the infected host Sf9 cells, with the N-terminus and C-terminus in the proper orientation. Affinity chromatography was used for the partial purification of the olp4 polypeptide from infected Sf9 cells. The identification and purification of this expressed olfactory receptor polypeptide could open the way for further characterization and functional studies of the olfactory receptor superfamily members.
Insights
Researchers identified and expressed a novel olfactory receptor (olp4) in vitro and in vivo. This work facilitates further functional studies of olfactory receptors.
Area of Science:
- Molecular Biology
- Neuroscience
- Biochemistry
Background:
- The olfactory receptor superfamily plays a crucial role in smell perception.
- Understanding the structure and function of individual olfactory receptors is essential for deciphering olfaction.
Purpose of the Study:
- To identify and characterize a new olfactory receptor clone (olp4) from a rat olfactory epithelium cDNA library.
- To express and analyze the olp4 protein in vitro and in vivo to understand its membrane insertion and localization.
Main Methods:
- Screening of a rat olfactory epithelium cDNA library.
- DNA sequencing and analysis of the olp4 clone.
- In vitro translation and immunoprecipitation using epitope tags and specific antibodies.
- Expression in Sf9 insect cells using the baculovirus system.
- Fluorescence cytometry and immunohistochemistry for cell surface localization.
- Affinity chromatography for protein purification.
Main Results:
- A novel olfactory receptor cDNA, olp4, was identified and sequenced.
- In vitro expression yielded a 27-29 kDa olp4 polypeptide, inserting into microsomal membranes.
- In vivo expression in Sf9 cells produced a 27-29 kDa polypeptide and a 34 kDa glycosylated form, localized to the cell surface.
Conclusions:
- The successful expression, membrane insertion, and cell surface localization of the olp4 receptor have been demonstrated.
- Partial purification of the expressed olp4 polypeptide was achieved.
- This study provides a foundation for further characterization and functional studies of olfactory receptors.