Abstract
An Escherichia coli expression system has been developed for pea cytosolic ascorbate peroxidase (APX). The enzyme was expressed as a fusion product with the E. coli maltose-binding protein for rapid, affinity chromatography purification. Recombinant ascorbate peroxidase (rAPX) was purified by tryptic digestion to separate the maltose-binding protein from rAPX followed by three chromatographic steps. The purified rAPX protein demonstrated identical electrophoretic, enzymatic, and spectral properties when compared to native APX isolated from pea shoots. Upon addition of an equal molar amount of H2O2, rAPX exhibits an initial decrease in the Soret maximum, which slowly converts to a stable, red-shifted Soret peak similar to that observed for cytochrome c peroxidase Compound I, indicating that rAPX Compound I consists of an oxyferryl (Fe(4+)-O) center. rAPX has been crystallized in a form suitable for crystal structure determination, and a preliminary set of native data to 2.6 A have been collected.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
