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Title:Cloning, High-Level Expression, and Characterization of Escherichia Coli Cytochrome B(562)
Author(s):Nikkila, Heli Paivikki
Doctoral Committee Chair(s):Sligar, Stephen G.
Department / Program:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Chemistry, Biochemistry
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.
Issue Date:1988
Description:109 p.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1988.
Other Identifier(s):(UMI)AAI8908789
Date Available in IDEALS:2014-12-15
Date Deposited:1988

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