Purification and characterization of a protein phosphatase (PP1-Arch) from the archaebacterium Sulfolobus solfataricus, isolation and expression of its gene

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dc.contributor.authorLeng, Jieen
dc.contributor.committeechairKennelly, Peter J.en
dc.contributor.committeememberBender, Patrick K.en
dc.contributor.committeememberBunce, George E.en
dc.contributor.committeememberSitz, Thomas O.en
dc.contributor.committeememberLee, John C.en
dc.contributor.departmentBiochemistry and Anaerobic Microbiologyen
dc.date.accessioned2014-03-14T21:17:41Zen
dc.date.adate2006-08-14en
dc.date.available2014-03-14T21:17:41Zen
dc.date.issued1994-11-05en
dc.date.rdate2006-08-14en
dc.date.sdate2006-08-14en
dc.description.abstractPP1-Arch was verified as a protein phosphatase by both acid molybdate extraction and thin layer electrophoresis. Soluble fraction was prepared from <i>Sulfolobus solfataricus</i>, from which PP1-Arch was purified over 1OOO-fold by DE-52 ion-exchange, hydroxyapatite, gel filtration (G- 100), and Mono Q FPLC chromatography. PP1-Arch was identified from the final purified sample by renaturation on an SDS-polyacrylamide gel. The molecular size of PP1-Arch was determined by both gel filtration chromatography and SDS-PAGE as 28 kDa and 33 kDa, respectively, which suggests that PP1-Arch is a monomer. PP1-Arch was found stable at temperatures as high as 90°C. Activation constants for the divalent metal ions Mn²⁺ and Ni²⁺, and the K<sub>m</sub> for phosphocasein were determined. Myosin light chain was found to be a substrate for PP1-Arch <i>in vitro</i>. EDTA, Cu²⁺, Zn²⁺, P<sub>i</sub>' and PP<sub>i</sub> were shown to be inhibitors of PP1-Arch, while many compounds known to affect eukaryotic protein phosphatase activities were found to be without noticeable effect. N-terminal and an internal peptide sequence of the enzyme were obtained. The gene for PP1-Arch was cloned by a combination of "touchdown" PCR and conventional cloning techniques. The PP1-Arch gene was sequenced on both strands, and the sequence was compared with ones from eukaryotes and bacteriophage λ. The sequence homology between PP1-Arch and PP1/PP2A/PP2B suggests that they belongs to the same genetic family. A recombinant plasmid which was derived from pT7-7 was constructed for expression of PP1-Arch. The PP1-Arch gene was expressed in <i>E. coli</i> and the activity of the expressed enzyme was tested and shown to be divalent metal ion-dependent. Formation of inclusion bodies of expressed PP1-Arch was demonstrated.en
dc.description.degreePh. D.en
dc.format.extentx, 117 leavesen
dc.format.mediumBTDen
dc.format.mimetypeapplication/pdfen
dc.identifier.otheretd-08142006-110059en
dc.identifier.sourceurlhttp://scholar.lib.vt.edu/theses/available/etd-08142006-110059/en
dc.identifier.urihttp://hdl.handle.net/10919/39141en
dc.language.isoenen
dc.publisherVirginia Techen
dc.relation.haspartLD5655.V856_1994.L464.pdfen
dc.relation.isformatofOCLC# 32749882en
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subject.lccLD5655.V856 1994.L464en
dc.subject.lcshArchaebacteriaen
dc.subject.lcshPhosphoprotein phosphatases -- Purificationen
dc.titlePurification and characterization of a protein phosphatase (PP1-Arch) from the archaebacterium Sulfolobus solfataricus, isolation and expression of its geneen
dc.typeDissertationen
dc.type.dcmitypeTexten
thesis.degree.disciplineBiochemistry and Anaerobic Microbiologyen
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen
thesis.degree.leveldoctoralen
thesis.degree.namePh. D.en

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