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Abstract
Rationally designed muteins of Candida albicans glucosamine-6-phosphate synthase, an enzyme known as a promising target for antifungal chemotherapy, were constructed, overexpressed in Escherichia coli and purified to near homogeneity. To facilitate and to optimize the purification of the enzyme, three recombinant versionscontaining internal oligoHis fragments were constructed: (i) by substituting residues 343 - 348 of the interdomain undecapeptide linker with hexaHis, (ii) by replacing solvent-exposed residues 655 - 660 of the isomerase domain with hexaHis, and (iii) by replacing amino acids at positions 568 and 569 with His residues to generate the three-dimensional hexaHis microdomain in the enzyme quaternary structure. The resulting constructs were effectively purified to nearhomogeneity by rapid, one-step immobilized metal-ion affinity chromatography and demonstrated activity and catalytic properties comparable with that of the wild-type enzyme. The construct containing the 655 - 660 hexaHis insert was found to be a homodimeric protein, which is the fi rst reported example of such quaternary structure of glucosamine-6-phosphate synthase of eukaryotic origin.
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- Category:
- Articles
- Type:
- artykuł w czasopiśmie wyróżnionym w JCR
- Published in:
-
JOURNAL OF MOLECULAR RECOGNITION
no. 25,
pages 564 - 570,
ISSN: 0952-3499 - Language:
- English
- Publication year:
- 2012
- Bibliographic description:
- Czarnecka J., Kwiatkowska K., Gabriel I., Wojciechowski M., Milewski S.: Engineering Candida albicans glucosamine-6-phosphate synthase for efficient enzyme purification// JOURNAL OF MOLECULAR RECOGNITION. -Vol. 25, nr. iss. 11 (2012), s.564-570
- Verified by:
- Gdańsk University of Technology
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