Type

Journal Article

Authors

Sean Doyle
Kevin A. Kavanagh
Emer P. Reeves
Imelda Vickers

Subjects

Microbiology

Topics
hydrogen ion concentration enzymology cloning molecular escherichia coli inclusion bodies binding sites immunoglobulin g larva pepstatin animals microbiology classification enzyme activation reverse transcriptase polymerase chain reaction secretion aspergillus fumigatus phylogeny recombinant proteins gene expression evolution molecular models biological molecular sequence data immunology amino acid sequence chemistry pathogenicity antagonists inhibitors moths pepstatins aspartic acid endopeptidases protein sorting signals genetics chromatography affinity metabolism enzyme linked immunosorbent assay isolation purification protein folding genes fungal hydrolysis base sequence culture media amino acid motifs virulence pharmacology baculoviridae survival rate

Isolation, activity and immunological characterisation of a secreted aspartic protease, CtsD, from Aspergillus fumigatus. (2006)

Abstract Aspergillus fumigatus is an opportunistic fungal pathogen that infects immunocompromised patients. A putative aspartic protease gene (ctsD; 1425 bp; intron-free) was identified and cloned. CtsD is evolutionarily distinct from all previously identified 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 purified, refolded, recombinant CtsD resulted in protease activation with a pH(opt)4.0 and specific activity=10 U/mg. Pepstatin A also inhibited recombinant CtsD activity by up to 72% thereby confirming classification as an aspartic protease. Native CtsD was also immunologically identified in culture supernatants and purified from fungal cultures using pepstatin-agarose affinity chromatography (7.8 microg CtsD/g mycelia). In A. fumigatus, semi-quantitative RT-PCR 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.
Collections Ireland -> Maynooth University -> PubMed

Full list of authors on original publication

Sean Doyle, Kevin A. Kavanagh, Emer P. Reeves, Imelda Vickers

Experts in our system

1
Sean Doyle
Maynooth University
Total Publications: 130
 
2
Kevin Kavanagh
Maynooth University
Total Publications: 183
 
3
Emer P Reeves
Royal College of Surgeons in Ireland
Total Publications: 63