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Large-scale production and characterization of an engineered human olfactory receptor

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dc.contributor.advisor Shuguang Zhang and Angela M. Belcher. en_US
dc.contributor.author Cook, Brian Lee en_US
dc.contributor.other Massachusetts Institute of Technology. Dept. of Biological Engineering. en_US
dc.date.accessioned 2011-02-23T14:31:34Z
dc.date.available 2011-02-23T14:31:34Z
dc.date.copyright 2008 en_US
dc.date.issued 2009 en_US
dc.identifier.uri http://hdl.handle.net/1721.1/61220
dc.description Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, February 2009. en_US
dc.description Cataloged from PDF version of thesis. en_US
dc.description Includes bibliographical references. en_US
dc.description.abstract Animal noses have evolved the ability to rapidly detect a seemingly infinite array of odors at minute concentrations. The basis of this sensitivity are the olfactory (smell) receptors - a large, highly related class of sensory G-protein coupled receptors that function together combinatorially to allow discrimination between a wide range of volatile and soluble molecules. However, the structural and functional mechanisms of these amazing receptors are not currently known. In order to begin to investigate the molecular mechanism(s) of olfaction, I have developed a mammalian expression system for the large-scale production and purification of functional olfactory receptor (OR) proteins in milligram quantities. Expressed OR genes were fabricated from scratch using PCR-based gene synthesis, which facilitated codon optimization and attachment of different affinity tags for purification. Established methods for the production and purification of rhodopsin were adapted to olfactory receptors through extensive optimization (including a full-spectrum screening of over 45 detergents). Key to the efficient extraction and solubilization of olfactory receptors tested is the use of novel zwitter-ionic fos-choline detergents. Following initial experiments on the inducible expression of a human olfactory receptor (hOR17-4) in adherent HEK293S cell cultures, the system was successfully scaled up using a suspension bioreactor. Large-scale culture allowed the purification of >10 milligrams of hOR17-4 monomer at >90%, which was suitable for subsequent X-ray crystallization screening trials. The purified protein was also characterized using several spectroscopic methods and shown to possess the correct secondary structure and several predicted post-translational modifications. To assay the functionality of purified (nonmembrane- bound) hOR17-4, we successfully developed an in vitro assay method using surface plasmon resonance (SPR) to demonstrate that the receptor retains functional selectivity in binding specific odorant ligands in a concentration-dependent manner. The application of these techniques to other olfactory receptors already shows promise and could lead to a generalized method for obtaining large quantities of any olfactory receptor in a rapid and simple manner. Such methods could prove extremely useful in elucidating the structural and functional mechanism(s) of olfactory receptors and in their integration into OR-based biosensor devices. en_US
dc.description.statementofresponsibility by Brian Lee Cook. en_US
dc.format.extent 165 p. en_US
dc.language.iso eng en_US
dc.publisher Massachusetts Institute of Technology en_US
dc.rights M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission. en_US
dc.rights.uri http://dspace.mit.edu/handle/1721.1/7582 en_US
dc.subject Biological Engineering. en_US
dc.title Large-scale production and characterization of an engineered human olfactory receptor en_US
dc.title.alternative Engineered human olfactory receptor en_US
dc.type Thesis en_US
dc.description.degree Ph.D. en_US
dc.contributor.department Massachusetts Institute of Technology. Dept. of Biological Engineering. en_US
dc.identifier.oclc 701378444 en_US


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