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Engineering Candida albicans glucosamine‐6‐phosphate synthase for efficient enzyme purification
Author(s) -
Czarnecka Justyna,
Kwiatkowska Karolina,
Gabriel Iwona,
Wojciechowski Marek,
Milewski Sławomir
Publication year - 2012
Publication title -
journal of molecular recognition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.401
H-Index - 79
eISSN - 1099-1352
pISSN - 0952-3499
DOI - 10.1002/jmr.2175
Subject(s) - enzyme , glucosamine , chemistry , linker , biochemistry , candida albicans , atp synthase , escherichia coli , recombinant dna , isomerase , affinity chromatography , protein engineering , phosphate , stereochemistry , biology , microbiology and biotechnology , computer science , gene , operating system
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 versions containing 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 near homogeneity 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 first reported example of such quaternary structure of glucosamine‐6‐phosphate synthase of eukaryotic origin. Copyright © 2012 John Wiley & Sons, Ltd.