Cloning, High-Level Expression, and Characterization of Escherichia Coli Cytochrome B(562)

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.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biochemistry

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2014

Author dc:creator

• Nikkila, Heli Paivikki

Contributors dc:contributor

• Sligar, Stephen G.

• Chemistry, Biochemistry

Identifier

(UMI)AAI8908789

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/70578