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Rickey Y. Yada - One of the best experts on this subject based on the ideXlab platform.
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structure function characterization of the recombinant Aspartic Proteinase a1 from arabidopsis thaliana
Phytochemistry, 2010Co-Authors: Miguel A Mazorramanzano, Takuji Tanaka, Rickey Y. YadaAbstract:Abstract Aspartic Proteinases (APs) are involved in several physiological processes in plants, including protein processing, senescence, and stress response and share many structural and functional features with mammalian and microbial APs. The heterodimeric Aspartic Proteinase A1 from Arabidopsis thaliana (AtAP A1) was the first acid protease identified in this model plant, however, little information exists regarding its structure function characteristics. Circular dichroism analysis indicated that recombinant AtAP A1 contained an higher α-helical content than most APs which was attributed to the presence of a sequence known as the plant specific insert in the mature enzyme. rAtAP A1 was stable over a broad pH range (pH 3–8) with the highest stability at pH 5–6, where 70–80% of the activity was retained after 1 month at 37 °C. Using calorimetry, a melting point of 79.6 °C was observed at pH 5.3. Cleavage profiles of insulin β-chain indicated that the enzyme exhibited a higher specificity as compared to other plant APs, with a high preference for the Leu 15 –Tyr 16 peptide bond. Molecular modeling of AtAP A1 indicated that exposed histidine residues and their interaction with nearby charged groups may explain the pH stability of rAtAP A1.
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An Investigation Of Gastric-like Aspartic Proteinase Molecular Chimeras
Biophysical Journal, 2009Co-Authors: Charity L. Parr, Rickey Y. YadaAbstract:Proplasmepsin II (zPMII) represents a structurally unique member of the Aspartic Proteinase family, with a prosegment-enzyme interaction that has never been reported. It has been a generally accepted assertion that the prosegment in pepsin-like Aspartic Proteinases is critical to Aspartic Proteinase folding, and to investigate this further two chimeric proteins were generated, one with the pepsinogen prosegment fused to the mature region of plasmepsin II (PMII) (pepproPMII) and a second with the prosegment of PMII fused to pepsin (PMIIpropep). Both chimeras were expressed from E. coli, however, PMIIpropep was extremely unstable and was rapidly degraded by trypsin, suggesting protein misfolding. Since a stable enzyme could not be generated PMIIpropep was not further studied. Alternatively, pepproPMII was capable of both autoactivation and synthetic substrate cleavage. In addition, both the zymogen and mature form of the enzyme had the same predicted secondary structures, suggesting that altering the PMII prosegment did not affect this level of protein conformation although the prosegment may play a role in enzyme stability. DSC and CD measurements indicated that pepproPMII had reduced thermal stability as compared to zPMII. It is proposed that this reduction of temperature stability resulted from the loss of the ability of the prosegment in PMII to stabilize the C-terminal domain of the enzyme. The ability of PMII to fold in the presence of a completely non-homologous prosegment suggests that the prosegment in gastric-like Aspartic Proteinases is not always critical to enzyme folding but likely plays a role in protein stabilization.
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expression and characterization of the recombinant Aspartic Proteinase a1 from arabidopsis thaliana
Phytochemistry, 2008Co-Authors: Miguel A Mazorramanzano, Rickey Y. YadaAbstract:Abstract The present study reports the recombinant expression, purification, and partial characterization of a typical Aspartic Proteinase from Arabidopsis thaliana (AtAP A1). The cDNA encoding the precursor of AtAP A1 was expressed as a functional protein using the yeast Pichia pastoris . The mature form of the rAtAP A1 was found to be a heterodimeric glycosylated protein with a molecular mass of 47 kDa consisting of heavy and light chain components, approx. 32 and 16 kDa, respectively, linked by disulfide bonds. Glycosylation occurred via the plant specific insert in the light chain. The catalytic properties of the rAtAP A1 were similar to other plant Aspartic Proteinases with activity in acid pH range, maximal activity at pH 4.0, K m of 44 μM, and k cat of 55 s −1 using a synthetic substrate. The enzyme was inhibited by pepstatin A.
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Construction, expression and characterization of a chimaeric mammalian-plant Aspartic Proteinase.
Biochemical Journal, 2003Co-Authors: Kenneth G. Payie, Takuji Tanaka, Rickey Y. YadaAbstract:: Aspartic Proteinases are a well-characterized class of Proteinases. In plants, all nascent Aspartic Proteinases possess a 100-amino-acid, plant-specific sequence (PSS) within their C-terminal lobe, presumed to possess a targeting role in vivo. In this study, the PSS domain from the Arabidopsis thaliana Aspartic Proteinase was inserted into porcine pepsinogen at the identical location found in nascent plant Aspartic Proteinases, to create a chimaeric mammalian-plant enzyme. Based on enzymic activity, this chimaeric enzyme demonstrated increases in pH stability above 6 and temperature stability above 60 degrees C compared with commercial pepsin. Differential scanning calorimetry of the chimaeric enzyme illustrated an approx. 2 degrees C increase in denaturation temperature ( T (m)), with increases in co-operativity and similar enthalpy values. Kinetic analysis indicated an increase in K (m) and decreased k (cat) compared with pepsin, but with a catalytic efficiency similar to the monomeric plant Aspartic Proteinase from wheat. Using oxidized insulin B-chain, the chimaeric enzyme demonstrated more restricted substrate specificity in comparison with commercial pepsin. This study highlights the use of a chimaeric enzyme strategy in order to characterize unique protein domains within enzyme families, and, for the first time, a putative structure-function role for the PSS as it pertains to plant Aspartic Proteinases.
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Purification, N-terminal sequencing and partial characterization of a novel Aspartic Proteinase from the leaves of Medicago sativa L. (alfalfa)
Biotechnology Letters, 2000Co-Authors: Kenneth G. Payie, Takuji Tanaka, Joel T. Weadge, Rickey Y. YadaAbstract:A novel Aspartic Proteinase (EC 3.4.23) from Medicago sativa L. (alfalfa) was purified to homogeneity using Source Q ion-exchange, concanavalin-A Sepharose and pepstatin-A agarose affinity chromatography. The enzyme, M _r=33.5 kDa, is monomeric and catalyzes the cleavage of a broad spectrum of peptide bonds of hydrophobic amino acids from pH 2.6 to 6.4. The enzyme is inhibited by pepstatin-A and is consistent with the properties of an Aspartic Proteinase. The N -terminal amino acid sequence of the protein shows 50 and 40% similarity with the cyprosin and barley Aspartic Proteinases, respectively.
Jukka Kervinen - One of the best experts on this subject based on the ideXlab platform.
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crystal structure of plant Aspartic Proteinase prophytepsin inactivation and vacuolar targeting
The EMBO Journal, 1999Co-Authors: Jukka Kervinen, Gregory J Tobin, Julia Costa, David S Waugh, Alexander Wlodawer, Alexander ZdanovAbstract:We determined at 2.3 A resolution the crystal structure of prophytepsin, a zymogen of a barley vacuolar Aspartic Proteinase. In addition to the classical pepsin‐like bilobal main body of phytepsin, we also traced most of the propeptide, as well as an independent plant‐specific domain, never before described in structural terms. The structure revealed that, in addition to the propeptide, 13 N‐terminal residues of the mature phytepsin are essential for inactivation of the enzyme. Comparison of the plant‐specific domain with NK‐lysin indicates that these two saposin‐like structures are closely related, suggesting that all saposins and saposin‐like domains share a common topology. Structural analysis of prophytepsin led to the identification of a putative membrane receptor‐binding site involved in Golgi‐mediated transport to vacuoles.
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transport and activation of the vacuolar Aspartic Proteinase phytepsin in barley hordeum vulgare l
Journal of Biological Chemistry, 1998Co-Authors: Stefanie Glathe, Jukka Kervinen, Gregory J Tobin, Alexander Wlodawer, Manfred Nimtz, Grace H Li, Terry D Copeland, David A Ashford, Julia CostaAbstract:Abstract The primary translation product of barley Aspartic Proteinase, phytepsin (EC 3.4.23.40), consists of a signal sequence, a propart, and mature enzyme forms. Here, we describe post-translational processing and activation of phytepsin during its transport to the vacuole in roots, as detected by using metabolic labeling and immunoprecipitation. After removal of the signal sequence, the glycosylated precursor of 53 kDa (P53) was produced and further processed to polypeptides of 31 and 15 kDa (P31 + P15) and, subsequently, to polypeptides of 26 and 9 kDa (P26 + P9), 45 min and 24 h after synthesis, respectively. The processing occurred in a late-Golgi compartment or post-Golgi compartment, because brefeldin A inhibited the processing, and P53 acquired partial endoglycosidase H resistance 30 min after synthesis, whereas P15 was completely resistant. The N-glycosylation inhibitor tunicamycin had no effect on transport, but the absence of glycans on P53 accelerated the proteolytic processing. Phytepsin was also expressed in baculovirus-infected insect cells. The recombinant prophytepsin underwent autoproteolytic activation in vitro and showed enzymatic properties similar to the enzyme purified from grains. However, a comparison of the in vitro/in vivoprocessing sites revealed slight differences, indicating that additional proteases are needed for the completion of the maturationin vivo.
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tissue specific localization of Aspartic Proteinase in developing and germinating barley grains
Planta, 1994Co-Authors: Kirsi Tormakangas, Jukka Kervinen, Anne Ostman, Teemu H TeeriAbstract:Resting seeds of several plant species, including barley grains, have been reported to contain Aspartic Proteinase (EC 3.4.23) activity. Here, the expression of the Hordeum vulgare L. Aspartic Proteinase (HvAP) was studied in developing and germinating grains by activity measurements as well as by immunocytochemical and in-situ hybridization techniques. Southern blotting suggests the presence of one to two HvAP-encoding genes in the barley genome, while Northern analysis reveals a single 2.1-kb mRNA in grains and vegetative tissues. Western blotting with antibodies to HvAP shows the same subunit structure in different grain parts. In developing grains, HvAP is produced in the embryo, aleurone layer, testa and pericarp, but in the starchy endosperm HvAP is present only in the crushed and depleted area adjacent to the scutellum. During seed maturation, HvAP-encoding mRNA remains in the aleurone layer and in the embryo, but the enzyme disappears from the aleurone cells. The enzyme, however, remains in the degenerating tissues of the testa and pericarp as well as in resting embryo and scutellum. During the first three days of germination, the enzyme reappears in the aleurone layer cells but is not secreted into the starchy endosperm. The HvAP is also expressed in the flowers, stem, leaves, and roots of barley. The wide localization of HvAP in diverse tissues suggests that it may have several functions appropriate to the needs of different tissues.
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comparative modelling of barley grain Aspartic Proteinase a structural rationale for observed hydrolytic specificity
FEBS Letters, 1994Co-Authors: Kunchur Guruprasad, Jukka Kervinen, Kirsi Tormakangas, T L BlundellAbstract:Abstract A model of the barley-grain Aspartic Proteinase (HvAP; Hordeum vulgare Aspartic Proteinase) has been constructed using the rule-based comparative modelling approach encoded in the COMPOSER suite of computer programs. The model was based on the high resolution crystal structures of six highly homologous Aspartic Proteinases. Results suggest that the overall three-dimensional structure of HvAP (excluding the plant-specific insert; 104 residues in HvAP) is closer to human cathepsin D than other Aspartic Proteinases of known three-dimensional structure. Comparisons of the complexes with the substrate modelled in the active site of HvAP with those of the same substrate modelled in the active site of other Aspartic Proteinases of known three-dimensional structure and specificity, define residues that may influence hydrolytic specificity of the barley enzyme. We have identified residues in the S4 (Ala12), S3 (Gln13, Thr111), S2 (Ala222, Thr287, Met289), S′1 and S′3 (Ile291), S′2 and S′3 (Gln74), S′2 (Arg295), and S′3 (Pro292) pockets, that may account for the observed trends in the kinetic behaviour and specificity when compared to other Aspartic Proteinases. The plant-specific inserted sequence, which may play a role in the transport of HvAP to plant vacuoles (lysosomes), is similar to the saposins and is predicted to be a mixed α-helical and β-strand domain.
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hydrolytic specificity of the barley grain Aspartic Proteinase
Phytochemistry, 1993Co-Authors: Jukka Kervinen, Paula Sarkkinen, Nisse Kalkkinen, Leena Mikola, Mart SaarmaAbstract:Abstract We recently published the primary structure and inhibition data of the barley grain Aspartic Proteinase (HvAP, Hordeum vulgare Aspartic Proteinase)
Miguel A Mazorramanzano - One of the best experts on this subject based on the ideXlab platform.
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structure function characterization of the recombinant Aspartic Proteinase a1 from arabidopsis thaliana
Phytochemistry, 2010Co-Authors: Miguel A Mazorramanzano, Takuji Tanaka, Rickey Y. YadaAbstract:Abstract Aspartic Proteinases (APs) are involved in several physiological processes in plants, including protein processing, senescence, and stress response and share many structural and functional features with mammalian and microbial APs. The heterodimeric Aspartic Proteinase A1 from Arabidopsis thaliana (AtAP A1) was the first acid protease identified in this model plant, however, little information exists regarding its structure function characteristics. Circular dichroism analysis indicated that recombinant AtAP A1 contained an higher α-helical content than most APs which was attributed to the presence of a sequence known as the plant specific insert in the mature enzyme. rAtAP A1 was stable over a broad pH range (pH 3–8) with the highest stability at pH 5–6, where 70–80% of the activity was retained after 1 month at 37 °C. Using calorimetry, a melting point of 79.6 °C was observed at pH 5.3. Cleavage profiles of insulin β-chain indicated that the enzyme exhibited a higher specificity as compared to other plant APs, with a high preference for the Leu 15 –Tyr 16 peptide bond. Molecular modeling of AtAP A1 indicated that exposed histidine residues and their interaction with nearby charged groups may explain the pH stability of rAtAP A1.
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expression and characterization of the recombinant Aspartic Proteinase a1 from arabidopsis thaliana
Phytochemistry, 2008Co-Authors: Miguel A Mazorramanzano, Rickey Y. YadaAbstract:Abstract The present study reports the recombinant expression, purification, and partial characterization of a typical Aspartic Proteinase from Arabidopsis thaliana (AtAP A1). The cDNA encoding the precursor of AtAP A1 was expressed as a functional protein using the yeast Pichia pastoris . The mature form of the rAtAP A1 was found to be a heterodimeric glycosylated protein with a molecular mass of 47 kDa consisting of heavy and light chain components, approx. 32 and 16 kDa, respectively, linked by disulfide bonds. Glycosylation occurred via the plant specific insert in the light chain. The catalytic properties of the rAtAP A1 were similar to other plant Aspartic Proteinases with activity in acid pH range, maximal activity at pH 4.0, K m of 44 μM, and k cat of 55 s −1 using a synthetic substrate. The enzyme was inhibited by pepstatin A.
Jan A Delcour - One of the best experts on this subject based on the ideXlab platform.
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a second Aspartic Proteinase associated with wheat gluten
Journal of Cereal Science, 2000Co-Authors: W Bleukx, Jan A DelcourAbstract:Abstract A second, distinct, Aspartic Proteinase (GlAP 2) associated with wheat gluten was partially purified by ammonium sulphate precipitation, anion exchange and hydrophobic interaction chromatography, a pH precipitation step and gel permeation chromatography. The enzyme has a molecular mass of c. 67 kDa and shows maximal hydrolytic activity towards haemoglobin at pH 3·0–3·5 and c. 50 °C. The enzyme is completely inhibited by pepstatin A and is less active towards haemoglobin than a previously purified gluten Aspartic Proteinase (GlAP). GlAP 2 is highly specific in its action towards oxidised insulin B-chain. This synthetic substrate is cleaved only at the Leu 15 -Tyr 16 position. The selectivity of GlAP 2 is therefore much higher than that of other plant APs. Furthermore, in contrast to the previously described GlAP, GlAP 2 shows almost no activity towards wheat gluten although it hydrolysed high molecular weight glutenin subunit 7. One Met-Ile peptide bond in the N-terminal domain and probably two Val-Leu peptide bonds in the repetitive domain of this subunit were cleaved by GlAP 2.
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specificity of a wheat gluten Aspartic Proteinase
Biochimica et Biophysica Acta, 1998Co-Authors: W Bleukx, Kristof Brijs, Sophie Torrekens, Fred Van Leuven, Jan A DelcourAbstract:Abstract The substrate and peptide bond specificities of a purified wheat gluten Aspartic Proteinase (GlAP) are studied. GlAP shows maximum gluten hydrolysing activity at pH 3.0. At this pH, especially the wheat high molecular weight glutenin subunits (HMW-GS) and to a lesser extent the low molecular weight glutenin subunits and gliadins are hydrolysed. GlAP has no obvious effect on albumins and globulins. In its action on oxidised insulin B-chain, GlAP forms eight peptides and has high specificity for peptide bonds located between amino acid residues with large hydrophobic side chains (Leu, Phe, Tyr) but the peptide bond Glu 13 -Ala 14 is also hydrolysed. Although structurally quite similar to a barley Aspartic Proteinase, the peptide bond specificity of GlAP towards oxidised insulin B-chain resembles slightly more that of a cardoon Aspartic Proteinase, cardosin B. HMW-GS 7, purified from cultivar Galahad-77, is rapidly hydrolysed by GlAP. N-Terminal amino acid sequence data show that GlAP cleaves at least one Met-Ile peptide bond at the end of the N-terminal domain and two Val-Leu peptide bonds in the repetitive domain of HMW-GS 7.
Michel Monod - One of the best experts on this subject based on the ideXlab platform.
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molecular cloning of an extracellular Aspartic Proteinase from rhizopus microsporus and evidence for its expression during infection
Medical Mycology, 2002Co-Authors: Christoph Schoen, Michel Monod, Utz Reichard, Hartmut Kratzin, R RuchelAbstract:An extracellular Aspartic Proteinase (Rmap) from Rhizopus microsporus var. rhizopodiformis was detected in the culture supernatant of a fungal isolate from a case of rhinocerebral mucormycosis (case HA). The Proteinase was purified to near homogeneity by ion exchange and affinity chromatography on pepstatin agarose. Based on its N-terminus the RMAP gene was cloned and found to code for 388 amino acids. The preproenzyme has an aminoterminal leader sequence of 65 amino acids, whereas the mature enzyme consists of 323 amino acids. The deduced amino-acid sequence of the preproenzyme was 82% homologous to an extracellular Aspartic Proteinase of Rhizopus niveus. Low stringency Southern blot analysis of R. microsporus DNA suggested the presence of other homologous genes. Expression of Rmap in Pichia pastoris was achieved, and the recombinant enzyme was active in the yeast culture supernatant. Both enzyme preparations exhibited a similar optimum of activity in the pH 2·5 region. Furthermore, Rmap was shown to act...
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molecular cloning and targeted deletion of pep2 which encodes a novel Aspartic Proteinase from aspergillus fumigatus
International Journal of Medical Microbiology, 2000Co-Authors: R Ruchel, Utz Reichard, Garry T Cole, Michel MonodAbstract:An Aspartic Proteinase PEP2 [EC 3.4.23.25] was purified from a cell wall fraction of Aspergillus fumigatus. The enzyme, which showed a broad range of activity from pH 2.0 to 7.0 and migrated as a single band of 39 kDa in SDS-PAGE, was not detected in the culture supernatant. A specific gene probe was designed on the basis of the N-terminal sequence of the native protein, and the PEP2 genomic and cDNA were isolated from corresponding libraries. The deduced amino acid sequence of PEP2 consists of 398 amino acids. A signal sequence of 18 amino acids and a proregion of another 52 amino acids were identified. The mature protein consists of 328 amino acids which include the two DTG-motifs of the active site common to almost all pepsin-like enzymes. PEP2 showed a 64% identity with the vacuolar Proteinase A (PrA), of Saccharomyces cerevisiae, and an 88% identity with PEPE, an Aspartic Proteinase of Aspergillus niger. Recombinant PEP2 was over-expressed in Pichia pastoris and the active enzyme was secreted into the culture supernatant. Targeted deletion of PEP2 did not affect vegetative growth or cell and colony morphology. Identification of Proteinases, such as PEP2, which are apparently associated with the Aspergillus cell wall raises new interest in these molecules with respect to their possible function in the pathogenesis of invasive aspergillosis.
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differential regulation of sap8 and saps which encode two new members of the secreted Aspartic Proteinase family in candida albicans
Microbiology, 1998Co-Authors: Michel Monod, Bernhard Hube, Daniela Hess, Dominique SanglardAbstract:SUMMARY: Secreted Aspartic Proteinases (Saps) contribute to the virulence of Candida albicans in systemic animal models of infection. Seven genes encoding Saps (SAM-SAP7) have been identified to date but evidence suggested the existence of additional SAP genes. The screening of a C. albicans iZEMBL3 genomic library for the presence of other SAP genes was undertaken. Two new genes, SAP8 and SAPS, were isolated. The N-terminal amino acid sequence deduced from SAP8 downstream of a Kex2plike cleavage site corresponds to the N-terminal amino acid sequence of the 41 kDa Sap isolated and characterized previously. SAP8 mRNA was expressed preferentially in yeasts at 25 "C after 6 and 9 h growth in BSA-containing medium. SAPS encodes an Aspartic Proteinase with a Kex2pllike cleavage site and contains a putative glycophosphatidylinositol-anchor signal at the C-terminus. Although the SAPS gene product has not yet been isolated from cultures of C. albicans, transcripts of SAPS were observed preferentially in later growth phases when SAP8 expression had decreased.
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high resolution structure of the extracellular Aspartic Proteinase from candida tropicalis yeast
Biochemistry, 1997Co-Authors: Jindrich Symersky, Michel Monod, S I FoundlingAbstract:The crystal structure of the secreted Aspartic Proteinase from Candida tropicalis yeast (SAPT) has been determined to 1.8 A resolution. The classic Aspartic Proteinase bilobal structure and domain ...
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molecular cloning and sequencing of the gene encoding an extracellular Aspartic Proteinase from aspergillus fumigatus
Fems Microbiology Letters, 1995Co-Authors: Utz Reichard, Michel Monod, R RuchelAbstract:Oligonucleotide primers based on conserved regions of the aspergillopepsins (EC 3.4.23.18) were used to PCR amplify a 650 bp segment of the gene encoding the extracellular Aspartic Proteinase (PEP) from Aspergillus fumigatus. The segment was used as a probe for isolating and sequencing the gene from a genomic library of the fungus. Likewise the cDNA was amplified by reverse PCR, cloned and sequenced. The pep gene was found to consist of four exons encoding for 395 aa. The pre-proenzyme deduced has an N-terminal leader sequence of 70 aa preceding the sequence of the mature enzyme consisting of 325 aa with a calculated molecular mass of 34.4 kDa and an isoelectric point of 3.95. The N-terminal sequence of the mature enzyme matched the N-terminal aa sequence of PEP exactly. The nucleotide and the aa sequences of the pre-proenzyme were 70% and 71% homologous to the corresponding sequences of the aspergillopepsin from A. niger var. awamori. Southern analysis of digested genomic A. fumigatus DNA with a specific PCR probe suggested the presence of a single copy of the pep gene.