Expression and Purification o f a Secretory Human Liver Carboxylesterase

Hdl Handle:
http://hdl.handle.net/10675.2/552404
Title:
Expression and Purification o f a Secretory Human Liver Carboxylesterase
Authors:
Miller, Amanda D.
Abstract:
Serine-dependent carboxyiesterases (E.C.3.1.1.1) are found in a variety o f tissues with high activity detected in the liver. Carboxyiesterases (CaE) hydrolyze aliphatic and aromatic esters, and aromatic amides; and play an important role in the detoxification of xenobiotic chemicals that contain organophosphate (OP) compounds. The detoxifying ability of CaE is limited by its low concentration in serum where it encounters OP compounds. Studies in our laboratory have shown that a pRc/CM V-hCaE plasmid construct, stably integrated into 293T cells, expresses a hum an liver CaE in culture. However, the enzyme levels reached a steady state of expression. The goals of this study were to overexpress a functional human liver CaE from a recombinant cDNA in a human cell line, and isolate and purify the recombinant protein. To accomplish these goals, the codon for the terminal leucine (CTG) of the human liver CaE retention signal, HIEL (hisile- glu leu), was mutated to that of arginine (CGG). The terminal G o f the arginine codon was also mutated to C, to produce a unique ECOA11YL restriction site to aid in clone selection. The recombinant plasmid, pRc/CMV-mhCaE, was isolated and stably integrated into human 293T cells. Expression of the altered cDNA resulted in secretion of an active CaE to levels in the range of two to five enzyme units per ten milliliters of growth medium. Secretory CaE displayed isoelectric focusing patterns similar to those of the native enzyme with no observable changes in activity. The secreted enzyme was partially purified by hydrophobic interaction chromatography and cibacron blue affinity chromatography. Partial enzyme purification was achieved, and CaE retained a high level of enzymatic activity.
Affiliation:
Not Listed
Issue Date:
Sep-1998
URI:
http://hdl.handle.net/10675.2/552404
Additional Links:
http://ezproxy.gru.edu/login?url=http://search.proquest.com/docview/304463639?accountid=12365
Type:
Dissertation
Appears in Collections:
Theses and Dissertations

Full metadata record

DC FieldValue Language
dc.contributor.authorMiller, Amanda D.en
dc.date.accessioned2015-05-06T19:44:29Zen
dc.date.available2015-05-06T19:44:29Zen
dc.date.issued1998-09en
dc.identifier.urihttp://hdl.handle.net/10675.2/552404en
dc.description.abstractSerine-dependent carboxyiesterases (E.C.3.1.1.1) are found in a variety o f tissues with high activity detected in the liver. Carboxyiesterases (CaE) hydrolyze aliphatic and aromatic esters, and aromatic amides; and play an important role in the detoxification of xenobiotic chemicals that contain organophosphate (OP) compounds. The detoxifying ability of CaE is limited by its low concentration in serum where it encounters OP compounds. Studies in our laboratory have shown that a pRc/CM V-hCaE plasmid construct, stably integrated into 293T cells, expresses a hum an liver CaE in culture. However, the enzyme levels reached a steady state of expression. The goals of this study were to overexpress a functional human liver CaE from a recombinant cDNA in a human cell line, and isolate and purify the recombinant protein. To accomplish these goals, the codon for the terminal leucine (CTG) of the human liver CaE retention signal, HIEL (hisile- glu leu), was mutated to that of arginine (CGG). The terminal G o f the arginine codon was also mutated to C, to produce a unique ECOA11YL restriction site to aid in clone selection. The recombinant plasmid, pRc/CMV-mhCaE, was isolated and stably integrated into human 293T cells. Expression of the altered cDNA resulted in secretion of an active CaE to levels in the range of two to five enzyme units per ten milliliters of growth medium. Secretory CaE displayed isoelectric focusing patterns similar to those of the native enzyme with no observable changes in activity. The secreted enzyme was partially purified by hydrophobic interaction chromatography and cibacron blue affinity chromatography. Partial enzyme purification was achieved, and CaE retained a high level of enzymatic activity.en
dc.relation.urlhttp://ezproxy.gru.edu/login?url=http://search.proquest.com/docview/304463639?accountid=12365en
dc.rightsCopyright protected. Unauthorized reproduction or use beyond the exceptions granted by the Fair Use clause of U.S. Copyright law may violate federal law.en
dc.subjectCarboxylestraseen
dc.subjectOrganophosphateen
dc.subjectOximeen
dc.subjectCell Cultureen
dc.subjectProtein Purificationen
dc.titleExpression and Purification o f a Secretory Human Liver Carboxylesteraseen
dc.typeDissertationen
dc.contributor.departmentNot Listeden
dc.description.advisorLanclos, Kenneth D.en
dc.description.committeeLewis, Jill E.; Scott, David F.; Sprinkle, Terry J.; Stoming, Terrance A.en
dc.description.degreeDoctor of Philosophy (Ph.D.)en
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