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dc.contributor.authorKim, Laura Yaunheeen_US
dc.date.accessioned2013-01-17T21:33:55Z
dc.date.available2013-01-17T21:33:55Z
dc.date.issued2012-11-15en_US
dc.identifier.urihttp://hdl.handle.net/1853/45820
dc.description.abstractMembrane proteins are involved in a number of disease pathologies and thus comprise a large number of drug targets. Determination of the high-resolution three-dimensional structure is essential for rational drug design, but several hurdles need to be overcome, primarily the over-expression and purification of said membrane proteins. Human leukotriene C4 synthase (hLTC4S), an 18 kDa integral membrane protein localized in the outer nuclear membrane of eosinophils and basophils, catalyzes the conjugation of LTA4 and reduced glutathione to produce LTC4. LTC4 and its metabolites LTD4 and LTE4 are the cysteinyl leukotrienes implicated in bronchoconstriction and inflammation pathways. The focus of my project involves optimizing the over-expression and purification of hLTC4S, which was heterologously expressed in Schizosaccharomyces pombe, purified by immobilized affinity chromatography, and finally "polished" with a buffer exchange step to remove excess co-purified lipids. The optimized protocol yielded ~1 mg of ~90% homogenous, pure protein per liter of cell culture. The finalized purified protein can then be used for further investigation of two-dimensional crystals by electron crystallography with the overall goal of structure determination.en_US
dc.publisherGeorgia Institute of Technologyen_US
dc.subjectElectron crystallographyen_US
dc.subjectMembrane proteinen_US
dc.subjectOverexpressionen_US
dc.subjectPurificationen_US
dc.subjectTwo-dimensional crystallizationen_US
dc.subject.lcshElectron microscopy
dc.subject.lcshLeukotrienes
dc.subject.lcshLeukotrienes Synthesis
dc.subject.lcshMembrane proteins
dc.titleOptimization of over-expression and purification of human leukotriene C4 synthase mutant R104A for structure-function studies by two-dimensional crystallization and electron crystallographyen_US
dc.typeThesisen_US
dc.description.degreeMSen_US
dc.contributor.departmentBiologyen_US
dc.description.advisorCommittee Chair: Schmidt-Krey, Ingeborg; Committee Member: DiChristina, Thomas; Committee Member: Lieberman, Raquelen_US


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