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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. 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;
Subjects: Science & Engineering > Biology
Science & Engineering > Institute of Immunology
Item ID: 2176
Depositing User: Dr. Sean Doyle
Date Deposited: 11 Oct 2010 13:17
Journal or Publication Title: Protein Expression and Purification
Publisher: Elsevier
Refereed: Yes
URI:

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