High-pressure-assisted reconstitution of recombinant chloroperoxidase.
An expression vector containing a T7 promoter and an OmpA signal sequence followed by the DNA sequence of mature chloroperoxidase from the fungus Caldariomyces fumago has been transformed into Escherichia coli. This construct gave high-level expression of apochloroperoxidase when induced with isopropyl thiogalactopyranoside. The nonglycosylated apoenzyme was secreted into periplasmic space. The recombinant apochloroperoxidase was expressed at a level representing about 2% of the total cellular protein. Before conversion to holoenzyme, the apochloroperoxidase was denatured in 8 M urea and partially purified by DEAE chromatography. Maximum yields of holoenzyme were obtained when the denatured apochloroperoxidase, dissolved in a refolding buffer containing iron protoporphyrin IX, calcium ions, and oxidized glutathione, was subjected to high pressure (207 MPa) at -12 degrees C and then allowed to refold at atmospheric pressure and room temperature. The recombinant holoenzyme was characterized by absorption and CD spectroscopy and tested for halogenation and peroxidation activity. The yield of active holochloroperoxidase was about 5% when high-pressure treatment was used as part of the reconstitution process. In the absence of pressure treatment, holoenzyme was formed at about the 1% level. The holochloroperoxidase preparations which resulted from high-pressure treatment showed, upon return to atmospheric pressure, a considerably higher content of native-like secondary structure compared to the nonpressurized preparations. These experiments show that active recombinant chloroperoxidase molecules can be produced, and prove that glycosylation is not a mandatory requirement for chloroperoxidase refolding.
Zong
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Q
Department of Biochemistry, Roger Adams Laboratory, University of Illinois, Urbana 61801, USA.
Osmulski
P A
PA
Hager
L P
LP
D001051:Apoenzymes / Q000235:genetics / Q000378:metabolism*; D002711:Chloride Peroxidase / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D006418:Heme / Q000378:metabolism*; D006874:Hydrostatic Pressure; D003904:Mitosporic Fungi / Q000201:enzymology; D010957:Plasmids; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D014508:Urea / Q000494:pharmacology
D001051:Apoenzymes; D005656:Fungal Proteins; D011994:Recombinant Proteins; D006418:Heme; D014508:Urea; D002711:Chloride Peroxidase