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Title:Studies of the Expression of Chloroperoxidase: Isolation and Sequence of a Chloroperoxidase Cdna Clone (complementary-Dna)
Author(s):Axley, Milton John
Department / Program:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Chemistry, Biochemistry
Abstract:The fungus Caldariomyces fumago secretes the enzyme chloroperoxidase at levels exceeding 400 milligrams per liter of culture medium when fructose is provided as the carbon source. Harnessing the cellular machinery which produces chloroperoxidase could provide a powerful method for the production and secretion of heterologous gene products. This report details the isolation of a chloroperoxidase cDNA clone and the study of the mechanisms which control the expression of chloroperoxidase.
An oligonucleotide was synthesized, based on known peptide sequence, to be complementary to chloroperoxidase mRNA. The oligonucleotide was used as a primer of cDNA synthesis to enrich the resultant clone bank for the sequence of interest. A cDNA clone which encoded part of chloroperoxidase was isolated from this enriched clone bank. The sequence of the clone revealed the presence of a putative signal peptide involved in secretion of the enzyme.
The isolated cDNA clone provided a hybridization probe for analysis of chloroperoxidase mRNA levels. Chloroperoxidase protein levels were analyzed by enzymatic and immunologic methods. It was found that chloroperoxidase mRNA and protein levels are induced by the presence of fructose in the culture medium and repressed by the presence of glucose. Chloroperoxidase expression is controlled at the mRNA level, and expression is not due to the structure or function of the protein. Therefore, the mechanisms which control chloroperoxidase expression are inherent to the primary structure of the gene and can be isolated for the production of heterologous proteins.
An intracellular precursor of chloroperoxidase was discovered. The precursor was much larger than chloroperoxidase, and the size could not be accounted for by the putative signal peptide. It is proposed that this precursor contains glycosylation which is trimmed from the protein as it is shuttled between compartments of the cell.
Issue Date:1986
Description:125 p.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1986.
Other Identifier(s):(UMI)AAI8701429
Date Available in IDEALS:2014-12-15
Date Deposited:1986

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