{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/70578"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/70578","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Cloning, High-Level Expression, and Characterization of Escherichia Coli Cytochrome B(562)","abstract":"The gene for the soluble cytochrome b$\\sb{562}$ from Escherichia coli B was cloned on a SalI fragment containing the full length gene, including the promotor. The DNA sequence analysis of the gene revealed the presence of a leader sequence, with the characteristics of an E. coli signal sequence, in front of the coding sequences for the mature protein. Localization studies on the overexpressed protein revealed that cytochrome b$\\sb{562}$ is translocated to the periplasmic space. Similar experiments on cytochrome b$\\sb{562}$-M7L (a point mutant of cytochrome b$\\sb{562}$ in which one of the heme axial ligands, methionine, was replaced by leucine), that was created in a site directed manner, revealed that the mutant protein which is unable to bind heme is transported to the periplasm as efficiently as the wild type protein. Cytochrome b$\\sb{562}$ expression from the internal promotor in the SalI fragment was studied using a multicopy plasmid. These studies revealed that cytochrome b$\\sb{562}$ expression is under glucose repression. A high level expression system for cytochrome b$\\sb{562}$ was constructed where the protein expression is driven from the lac-promotor located on pUC18. The level of expression of cytochrome b$\\sb{562}$ in this system is 3-5% of total protein. The overexpressed protein was purified and characterized by various spectroscopic techniques. The spectral analysis and N-terminal sequence analysis of the purified protein shows it is identical to the soluble, chromosomally expressed cytochrome b$\\sb{562}$ earlier purified and characterized from Escherichia coli B.","abstract_html":"The gene for the soluble cytochrome b$\\sb{562}$ from Escherichia coli B was cloned on a SalI fragment containing the full length gene, including the promotor. The DNA sequence analysis of the gene revealed the presence of a leader sequence, with the characteristics of an E. coli signal sequence, in front of the coding sequences for the mature protein. Localization studies on the overexpressed protein revealed that cytochrome b$\\sb{562}$ is translocated to the periplasmic space. Similar experiments on cytochrome b$\\sb{562}$-M7L (a point mutant of cytochrome b$\\sb{562}$ in which one of the heme axial ligands, methionine, was replaced by leucine), that was created in a site directed manner, revealed that the mutant protein which is unable to bind heme is transported to the periplasm as efficiently as the wild type protein. Cytochrome b$\\sb{562}$ expression from the internal promotor in the SalI fragment was studied using a multicopy plasmid. These studies revealed that cytochrome b$\\sb{562}$ expression is under glucose repression. A high level expression system for cytochrome b$\\sb{562}$ was constructed where the protein expression is driven from the lac-promotor located on pUC18. The level of expression of cytochrome b$\\sb{562}$ in this system is 3-5% of total protein. The overexpressed protein was purified and characterized by various spectroscopic techniques. The spectral analysis and N-terminal sequence analysis of the purified protein shows it is identical to the soluble, chromosomally expressed cytochrome b$\\sb{562}$ earlier purified and characterized from Escherichia coli B.","abstract_has_math":true,"creators":["Nikkila, Heli Paivikki"],"institution":"University of Illinois at Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Biochemistry","degree_department":null,"school":null,"contributors":["Sligar, Stephen G."],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2014,"date_issued":"2014-12-15T23:44:13Z","date_published":"2014-12-15T23:44:13Z","updated_at":"2026-07-22T22:26:03Z","subjects":["Chemistry, Biochemistry"],"languages":[],"rights":[],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["(UMI)AAI8908789"],"render_values":[{"text":"(UMI)AAI8908789","href":null,"code":true}]}]},"links":{"outbound_url":"http://hdl.handle.net/2142/70578","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Sligar, Stephen G."]},{"key":"dc:creator","label":"Author","values":["Nikkila, Heli Paivikki"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2014-12-15T23:44:13Z","10000-01-01","1988"]},{"key":"dc:type","label":"Dc Type","values":["text"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Biochemistry"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph.D."]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["University of Illinois at Urbana-Champaign"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Chemistry, Biochemistry"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["http://hdl.handle.net/2142/70578","(UMI)AAI8908789"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["The gene for the soluble cytochrome b$\\sb{562}$ from Escherichia coli B was cloned on a SalI fragment containing the full length gene, including the promotor. The DNA sequence analysis of the gene revealed the presence of a leader sequence, with the characteristics of an E. coli signal sequence, in front of the coding sequences for the mature protein. Localization studies on the overexpressed protein revealed that cytochrome b$\\sb{562}$ is translocated to the periplasmic space. Similar experiments on cytochrome b$\\sb{562}$-M7L (a point mutant of cytochrome b$\\sb{562}$ in which one of the heme axial ligands, methionine, was replaced by leucine), that was created in a site directed manner, revealed that the mutant protein which is unable to bind heme is transported to the periplasm as efficiently as the wild type protein. Cytochrome b$\\sb{562}$ expression from the internal promotor in the SalI fragment was studied using a multicopy plasmid. These studies revealed that cytochrome b$\\sb{562}$ expression is under glucose repression. A high level expression system for cytochrome b$\\sb{562}$ was constructed where the protein expression is driven from the lac-promotor located on pUC18. The level of expression of cytochrome b$\\sb{562}$ in this system is 3-5% of total protein. The overexpressed protein was purified and characterized by various spectroscopic techniques. The spectral analysis and N-terminal sequence analysis of the purified protein shows it is identical to the soluble, chromosomally expressed cytochrome b$\\sb{562}$ earlier purified and characterized from Escherichia coli B.","Made available in DSpace on 2014-12-15T23:44:13Z (GMT). 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The DNA sequence analysis of the gene revealed the presence of a leader sequence, with the characteristics of an E. coli signal sequence, in front of the coding sequences for the mature protein. Localization studies on the overexpressed protein revealed that cytochrome b$\\sb{562}$ is translocated to the periplasmic space. Similar experiments on cytochrome b$\\sb{562}$-M7L (a point mutant of cytochrome b$\\sb{562}$ in which one of the heme axial ligands, methionine, was replaced by leucine), that was created in a site directed manner, revealed that the mutant protein which is unable to bind heme is transported to the periplasm as efficiently as the wild type protein. Cytochrome b$\\sb{562}$ expression from the internal promotor in the SalI fragment was studied using a multicopy plasmid. These studies revealed that cytochrome b$\\sb{562}$ expression is under glucose repression. A high level expression system for cytochrome b$\\sb{562}$ was constructed where the protein expression is driven from the lac-promotor located on pUC18. The level of expression of cytochrome b$\\sb{562}$ in this system is 3-5% of total protein. The overexpressed protein was purified and characterized by various spectroscopic techniques. The spectral analysis and N-terminal sequence analysis of the purified protein shows it is identical to the soluble, chromosomally expressed cytochrome b$\\sb{562}$ earlier purified and characterized from Escherichia coli B.","Made available in DSpace on 2014-12-15T23:44:13Z (GMT). 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