Isolation, activity and immunological characterisation of a secreted aspartic protease, CtsD, from Aspergillus fumigatus


Vickers, Imelda and Reeves, Emer P. and Kavanagh, Kevin A. and Doyle, Sean (2007) Isolation, activity and immunological characterisation of a secreted aspartic protease, CtsD, from Aspergillus fumigatus. Protein Expression and Purification, 53 (1). pp. 216-224. ISSN 1046-5928

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Abstract

Aspergillus fumigatus is an opportunistic fungal pathogen that infects immunocompromised patients. A putative aspartic protease gene (ctsD; 1425 bp; intron-free) was identiWed and cloned. CtsD is evolutionarily distinct from all previously identiWed A. fumigatus aspartic proteases. Recombinant CtsD was expressed in inclusion bodies in Escherichia coli (0.2mg/g cells) and subjected to extensive proteolysis in the baculovirus expression system. Activation studies performed on puriWed, refolded, recombinant CtsD resulted in protease activation with a pHopt4.0 and speciWc activityD10 U/mg. Pepstatin A also inhibited recombinant CtsD activity by up to 72% thereby conWrming classiWcation as an aspartic protease. Native CtsD was also immunologically identiWed in culture supernatants and puriWed from fungal cultures using pepstatin–agarose aYnity chromatography (7.8 g CtsD/g mycelia). In A. fumigatus, semi-quantitative RTPCR analysis revealed expression of ctsD in minimal and proteinaceous media only. Expression of ctsD was absent under nutrient-rich conditions. Expression of ctsD was also detected, in vivo, in the Galleria mellonella virulence model following A. fumigatus infection. © 2006 Elsevier Inc. All rights reserved.

Item Type: Article
Keywords: ELISA; Proteases; Virulence; Insect model; Siderophores; Pepstatin, Galleria mellonella;
Academic Unit: Faculty of Science and Engineering > Biology
Faculty of Science and Engineering > Research Institutes > Institute of Immunology
Item ID: 2176
Identification Number: 10.1016/j.pep.2006.12.012
Depositing User: Dr. Sean Doyle
Date Deposited: 11 Oct 2010 13:17
Journal or Publication Title: Protein Expression and Purification
Publisher: Elsevier
Refereed: Yes
Funders: Irish Research Council for Science Engineering and Technology (IRCSET), Irish Government Programme for Research in Third Level Institutions (PRTLI, Cycle 3), National Institute of Allergy and Infectious Disease, Wellcome Trust, Fondo de Investicagiones Sanitarias, Health Research Board (HRB)
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