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|Title:||Cloning, Sequencing and Expression of Chloroperoxidase Complementary-Dna in Escherichia Coli|
|Doctoral Committee Chair(s):||Hager, Lowell P.|
|Department / Program:||Biochemistry|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||Chloroperoxidase is an enzyme secreted by the filamentous fungus Caldariomyces fumago. The enzyme catalyzes peroxidative halogenation of a number of organic compounds. Halides used include chloride, bromide and iodide. It has been shown previously that the enzyme is functionally related to plant peroxidase and catalase. It is also spectroscopically related to the cytochromes P450. It is of fundamental importance to investigate the structure-function relationships of chloroperoxidase in order to understand these phenomena. This thesis describes the accomplishment of a crucial step toward this final goal.
The primary structure of chloroperoxidase has been determined by cDNA cloning and sequencing. Analysis of the sequence data revealed the following: The reading frame of the CPO mRNA encodes a protein of 321 amino acid residues. The 21 amino acid residues at the N-terminus of the predicted protein sequence has been proposed to be a signal peptide. This putative signal peptide has a positively charged lysine near the N-terminus, followed by a hydrophobic core sequence. The hydrophobic sequence has a predicted secondary structure of $\alpha$-helix. The mature sequence of chloroperoxidase is composed of 300 amino acid residues. The sequence contains three cysteine residues and three potential glycosylation sites.
Efforts were made to express enzymatically active chloroperoxidase in E. coli. Expression of a polypeptide was achieved that was capable of cross reacting with the affinity purified rabbit anti-CPO serum.
Some preliminary experiments were done to develop C. fumago as an expression system. A fragment which contains the cpo gene and flanking sequences was subcloned into the plasmid pUC18. The resulting plasmid has been named pCF6. Most of the CPO encoding sequence was subsequently deleted. The resulting plasmid has been designated pCF6D. The deletion plasmid, pCF6D, will be used to delete the endogenous cpo gene in C. fumago by gene replacement technique.
Finally, a detailed restriction map has been produced to facilitate the recombinant DNA manipulation of pCF6 and pCF6D.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1988.
|Date Available in IDEALS:||2014-12-15|