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Sligar, Stephen G. Nikkila, Heli Paivikki 2014-12-15T23:44:13Z 2014-12-15T23:44:13Z 10000-01-01 1988 1988 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). No. of bitstreams: 1 8908789.pdf: 2887586 bytes, checksum: 452a00583644b934d50000cf81be3935 (MD5) Previous issue date: 1988 Embargo set by: Seth Robbins for item 70744 Lift date: Forever Reason: Restricted to the U of I community idenfinitely during batch ingest of legacy ETDs Restricted to the U of I community idenfinitely during batch ingest of legacy ETDs U of I Only 109 p. Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1988. http://hdl.handle.net/2142/70578 (UMI)AAI8908789 Chemistry, Biochemistry Cloning, High-Level Expression, and Characterization of Escherichia Coli Cytochrome B(562) text Biochemistry Biochemistry University of Illinois at Urbana-Champaign Dissertation Ph.D.