{"id":{"repo_id":"vt","oai_identifier":"oai:vtechworks.lib.vt.edu:10919/39141"},"canonical_url":"https://search.dev.ndltd.org/etd/vt/oai:vtechworks.lib.vt.edu:10919/39141","repository":{"repo_id":"vt","name":"Virginia Tech","base_url":"https://vtechworks.lib.vt.edu/oai/request"},"display":{"title":"Purification and characterization of a protein phosphatase (PP1-Arch) from the archaebacterium Sulfolobus solfataricus, isolation and expression of its gene","abstract":"PP1-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.","abstract_html":"PP1-Arch was verified as a protein phosphatase by both acid molybdate extraction and thin layer electrophoresis. Soluble fraction was prepared from &lt;i&gt;Sulfolobus solfataricus&lt;/i&gt;, 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&lt;sub&gt;m&lt;/sub&gt; for phosphocasein were determined. Myosin light chain was found to be a substrate for PP1-Arch &lt;i&gt;in vitro&lt;/i&gt;. EDTA, Cu²⁺, Zn²⁺, P&lt;sub&gt;i&lt;/sub&gt;&#x27; and PP&lt;sub&gt;i&lt;/sub&gt; 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 &quot;touchdown&quot; 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 &lt;i&gt;E. coli&lt;/i&gt; 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.","abstract_has_math":false,"creators":["Leng, Jie"],"institution":"Virginia Tech","degree_name":"Ph. D.","degree_level":"doctoral","degree_discipline":"Biochemistry and Anaerobic Microbiology","degree_department":"Biochemistry and Anaerobic Microbiology","school":null,"contributors":[],"advisors":[],"committee_chairs":["Kennelly, Peter J."],"committee_members":["Bender, Patrick K.","Bunce, George E.","Sitz, Thomas O.","Lee, John C."],"year":1994,"date_issued":"1994-11-05","date_published":"1994-11-05","updated_at":"2026-07-22T22:20:17Z","subjects":[],"languages":["en"],"rights":["In Copyright"],"rights_urls":["http://rightsstatements.org/vocab/InC/1.0/"],"identifier_entries":[{"key":"dc:identifier.other","label":"Dc Identifier Other","values":["etd-08142006-110059"],"render_values":[{"text":"etd-08142006-110059","href":null,"code":true}]}]},"links":{"outbound_url":"http://hdl.handle.net/10919/39141","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.committeechair","label":"Committee Chair","values":["Kennelly, Peter J."]},{"key":"dc:contributor.committeemember","label":"Committee Member","values":["Bender, Patrick K.","Bunce, George E.","Sitz, Thomas O.","Lee, John C."]},{"key":"dc:contributor.department","label":"Department","values":["Biochemistry and Anaerobic Microbiology"]},{"key":"dc:creator","label":"Author","values":["Leng, Jie"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2014-03-14T21:17:41Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2014-03-14T21:17:41Z","2006-08-14"]},{"key":"dc:date.issued","label":"Date","values":["1994-11-05"]},{"key":"dc:publisher","label":"Institution","values":["Virginia Tech"]},{"key":"dc:type","label":"Dc Type","values":["Dissertation"]},{"key":"dc:type.dcmitype","label":"Dc Type Dcmitype","values":["Text"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Biochemistry and Anaerobic Microbiology"]},{"key":"thesis:degree_level","label":"Degree Level","values":["doctoral"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph. 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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."]},{"key":"dc:description.degree","label":"Dc Description Degree","values":["Ph. D."]},{"key":"dc:format.medium","label":"Dc Format Medium","values":["BTD"]},{"key":"dc:format.mimetype","label":"Dc Format Mimetype","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Purification and characterization of a protein phosphatase (PP1-Arch) from the archaebacterium Sulfolobus solfataricus, isolation and expression of its gene"]}]}],"canonical_facts":{"dc:contributor.committeechair":["Kennelly, Peter J."],"dc:contributor.committeemember":["Bender, Patrick K.","Bunce, George E.","Sitz, Thomas O.","Lee, John C."],"dc:contributor.department":["Biochemistry and Anaerobic Microbiology"],"dc:creator":["Leng, Jie"],"dc:date.accessioned":["2014-03-14T21:17:41Z"],"dc:date.available":["2014-03-14T21:17:41Z","2006-08-14"],"dc:date.issued":["1994-11-05"],"dc:description.abstract":["PP1-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."],"dc:description.degree":["Ph. D."],"dc:format.medium":["BTD"],"dc:format.mimetype":["application/pdf"],"dc:identifier.other":["etd-08142006-110059"],"dc:identifier.uri":["http://hdl.handle.net/10919/39141"],"dc:language.iso":["en"],"dc:publisher":["Virginia Tech"],"dc:rights":["In Copyright"],"dc:rights.uri":["http://rightsstatements.org/vocab/InC/1.0/"],"dc:title":["Purification and characterization of a protein phosphatase (PP1-Arch) from the archaebacterium Sulfolobus solfataricus, isolation and expression of its gene"],"dc:type":["Dissertation"],"dc:type.dcmitype":["Text"],"thesis:degree_discipline":["Biochemistry and Anaerobic Microbiology"],"thesis:degree_level":["doctoral"],"thesis:degree_name":["Ph. D."],"thesis:institution_name":["Virginia Polytechnic Institute and State University"]},"updated_at":"2026-07-22T22:20:17Z"}