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Isolation of recombinant cysteine dioxygenase protein from Trichophyton mentagrophytes
Author(s) -
Kasperova Alena,
Kunert Jiri,
Horynova Milada,
Weigl Evzen,
Sebela Marek,
Lenobel Rene,
Raska Milan
Publication year - 2011
Publication title -
mycoses
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.13
H-Index - 69
eISSN - 1439-0507
pISSN - 0933-7407
DOI - 10.1111/j.1439-0507.2010.01948.x
Subject(s) - trichophyton , recombinant dna , isolation (microbiology) , microbiology and biotechnology , cysteine , biology , chemistry , enzyme , biochemistry , gene , antifungal
Summary Cysteine dioxygenase (CDO, EC 1.13.11.20) catalyses the oxygenation of cysteine to cysteine sulphinic acid leading to the production of sulphite, sulphate and taurine as the final metabolites of cysteine catabolism. Keratinolytic fungi secrete sulphite and sulphate to reduce disulphide bridges in host tissue keratin proteins as the first step of keratinolysis. In the present study, we describe the identification of cDNA, as well as expression and characterisation of recombinant CDO protein from Trichophyton mentagrophytes . The cDNA was amplified using primers designed on the basis of high conservancy CDO regions identified in other fungi. PCR product was cloned and sequenced. Recombinant CDO was expressed in Escherichia coli , and affinity purified and identified by matrix‐assisted laser desorption/ionization – time‐of‐flight mass spectrometry (MALDI‐TOF MS). Enzyme activity was assayed by monitoring the production of cysteine sulphinate using mass spectrometry. The Cdo cDNA encodes for a protein consisting of 219 amino acids. Recombinant CDO protein C‐terminally fused with a His tag was purified by affinity chromatography. The CDO purified under native condition was proved to be enzymatically active. Protein identity was confirmed by MALDI‐TOF MS. Comparison of cDNA sequence with those identified in other fungi revealed significant homology. Identification of T. mentagrophytes CDO provides indispensable tools for future studies of dermatophyte pathogenicity and development of new approaches for prevention and therapy.