Cloning, High-Level Expression, and Characterization of Escherichia Coli Cytochrome B(562)
Nikkila, Heli Paivikki
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https://hdl.handle.net/2142/70578
Description
Title
Cloning, High-Level Expression, and Characterization of Escherichia Coli Cytochrome B(562)
Author(s)
Nikkila, Heli Paivikki
Issue Date
1988
Doctoral Committee Chair(s)
Sligar, Stephen G.
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(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.
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