0052 5.55/30.1 Proteolysis involved in cellular protein catabolic process Apoptosis inhibitor Bioinformatics analysis of TR TR was predicted as a secretory protein with the presence of signal sequences with good predictive value (signalP probability, 0.808). The
protein localization of TR was predicted using WoLF PSORT, and the result also indicated that this protein might be an extracellular protein (Query Protein WoLFPSORT prediction: extr, 12.0; cyto, 6.5; cyto_nucl, 4.0; mito, 3.0; pero, 2.0). This protein was BLAST-searched for sequence homology with human proteins and other fungi using the BLAST program LY2874455 (http://www.ncbi.nlm.nih.gov/BLASTp). The results indicated that TR of A. fumigatus had no matches with human proteins. Furthermore, TR of A. fumigatus had low homology with other fungi, such as Candida albicans (25%), C. tropicalis (25%),
C. glabrata (24%), C. guilliermondii (27%), C. dubliniensis (23%), Saccharomyces cerevisiae (24%), Cryptococcus neoformans (28%), and Penicillium marneffei (27%). This protein was also BLAST-searched for sequence homology with all protein databases using the Uniprot program (http://www.uniprot.org). The results indicated that TR of A. fumigatus has < 55% homology with all proteins in the databases, excluding pyridine nucleotide-disulphide oxidoreductase of A. fischeri (identitiy, 94%) and the putative uncharacterized protein of A. terreus (identitity, 80%). TR of A. fumigatus also had low homology with most other Aspergillus species, such as A. oryzae (55%), A. flavus buy GDC-0941 (54%), A. nidulans (50%), A. clavatus (47%), and A. niger (41%), as shown in Additional file 3. Expression and antigenicity Inositol oxygenase of TR recombinant protein After induction by isopropyl-β-D-thiogalactoside (IPTG), the recombinant
6-His-tagged TR was expressed, and a novel protein band corresponding to 36 kDa was detected by SDS-PAGE (Figure 3A). Most of the recombinant proteins were soluble. After purification using a TALON metal affinity resin, the protein purity was approximately 91%. Protein identity was unambiguously confirmed by MALDI-TOF MS, whereas following tryptic digestion proteins were identified yielding 37% sequence coverage (the MS spectra are shown in Additional file 4). Western blot showed that the recombinant proteins could be recognized by the sera from all six patients with proven IA (Figure 3B). Figure 3 SDS-PAGE and Western blot analysis of the recombinant thioredoxin reductase GliT (TR) of A. fumigatus. (A): SDS-PAGE analysis of the recombinant TR expressed in Escherichia coli BL21. Lane M, molecular weight marker; lane 1, pET28a -TR in E. coli BL21, 1 mM isopropyl-β- D – thiogalactoside induced for 5 h; lane 2, pET28a-TR in E. coli BL21, not induced; lane 3, purified recombinant TR; (B): Western blot analysis of the purified recombinant TR with sera of 6 patients with proven IA, pooled control patients, and monoclonal mouse anti-His antibody.