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https://hdl.handle.net/2142/84880
Description
Title
Expression of Recombinant Chloroperoxidase
Author(s)
Zong, Qin
Issue Date
1997
Doctoral Committee Chair(s)
Hager, Lowell P.
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
Language
eng
Abstract
Chloroperoxidase from the filamentous fungus Caldariomyces fumago is a heme protein with a unique structure and broad catalytic activities. In this thesis research, we report on the development of protein expression systems for chloroperoxidase. Both prokaryotic and eukaryotic expression systems were investigated. Chloroperoxidase was overexpressed in E. coli pET expression system. The expressed apoenzyme was secreted into periplasmic space at an expression level representing about 2% of the total cellular protein. The apochloroperoxidase in the insoluble inclusion bodies was solubilized in 8 M urea and partially reconstituted with iron protoporphyrin IX, then the partially reconstituted holochloroperoxidase was subjected to high pressure (207 MPa) treatment at $-12\sp\circ$C. The yield of active holochloroperoxidase was about 5% when reconstitution was carried out at high pressure. The pressurized holochloroperoxidase preparations showed a considerably higher content of native-like secondary structure compared to the nonpressurized samples. In order to express the chloroperoxidase as a fully modified and functional holoenzyme, eukaryotic expression systems including yeast Pichia pastoris expression system, baculovirus insect cell expression system and the filamentous fungus host, Caldariomyces fumago were investigated. The Caldariomyces fumago appears to be the best expression host. It is the only expression system can be used to produce fully modified and active recombinant chloroperoxidase. The expression of recombinant chloroperoxidase gene was placed under the control of a 900 bp chloroperoxidase promoter. A FLAG-epitope was fused in the N-terminal of the mature chloroperoxidase gene for identification and purification. The expressed FLAGCPO fusion protein was purified on the M2 FLAG-epitope affinity beads from the recombinant Caldariomyces fumago culture medium. The purified recombinant chloroperoxidase has both chlorination and peroxidation activities. This fungal homologous expression system should be suitable for expressing the recombinant chloroperoxidase for the site-directed and sequential random mutagenesis studies.
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