Identification and expression of Pen c 2, a novel allergen from Penicillium citrinum

Abstract

The mould genus, Penicillium, is known to be a significant source of environmental aero-allergens. One important allergen from Penicillium citrinum, Pen c 2, has been identified by means of two-dimensional immunoblotting using IgE-containing patients' sera. This novel allergen was cloned, sequenced and expressed in Escherichia coli. The cloned cDNA encodes a large 457-amino acid protein precursor containing a 16-amino acid signal peptide, a 120-amino acid propeptide and the 321-amino acid mature protein. Comparison of the Pen c 2 sequence with known protein sequences revealed shared high sequence similarities with two vacuolar serine proteases from Aspergillus niger and Saccharomyces cerevisiae. Asp-46, His-78 and Ser-244 were found to constitute the catalytic triad of the 39-kDa Pen c 2. The DNA coding for Pen c 2 was cloned into vector PQE-30 and expressed in E. coli as a His-tag fusion protein that bound serum IgE from Penicillium-allergic patients on immunoblots. Recombinant Pen c 2 could therefore be used effectively for diagnosis and also potentially for the treatment of mould-derived allergic disorders.

Identification and expression of an allergen Asp f 13 from Aspergillus fumigatus and epitope mapping using human IgE antibodies and rabbit polyclonal antibodies

“…Der p 1, which is a cysteine proteinase [32], and Der p 3, Der p 6, and Der p 9, which are serine proteases [33][34][35]. Moreover, we previously reported that the major allergens Pen c 1 and Pen c 2 in P. citrinum are also serine proteases [17,18]. The epithelium-detaching protease activity of allergens is currently under investigation.…”

“…The sense primer used was 5h-GCCTTGACCACTCAAAAGGGC-3h, coding for the seven N-terminal amino acids, and the antisense primer was 5h-TGA-GGGGTGGCCATGGAAGTA-3h for the active site region. To obtain the 5h and 3h portion of the Asp f 13 cDNA, the RACE PCR protocol was used as described previously [17]. The coding sequence of the Asp f 13 gene was thus amplified by PCR and the amplified product analysed by electrophoresis and subcloned into the pGEM-T vector (Promega).…”

“…After centrifugation at 12 000 g for 10 min at 4 mC, the supernatant was brought to a final concentration of 5 % trichloracetic acid and the resultant precipitate washed with cold diethyl ether, air dried, and stored at k70 mC. Dried powder (2 mg) was suspended in 100 µl of sample solution (2 % pharmalyte, 9n0 M urea, 0n5 % Triton X-100, 60 mM dithiothreitol, and 0n003 % bromphenol blue) and 2-DE performed as previously described [17]. The first dimension consisted of isoelectric focussing (IEF) with an immobilized pH gradient of 3-10 in a Multiphor II horizontal electrophoresis system (Pharmacia, Uppsala, Sweden), while the second dimension was SDS\PAGE.…”

Proteomics and Immunological Analysis of a Novel Shrimp Allergen, Pen m 2

“…The sense primer used was 5Ј-GCTGACGCTGCTGT(T/C)ATTGA(A/G)AAG-3Ј, encoding the eight N-terminal amino acids (ADAAVIEK), while the antisense primer was 5Ј-GCGGTC(G/A)TGGTG(A/T)GAGAA(A/G)GGAAT-3Ј, encoding a conserved sequence of amino acids (IPFSHHDR) found in arginine kinases. To obtain the 5Ј and 3Ј portions of the Pen m 2 cDNA, the RACE PCR protocol was used as described previously (26). The coding sequence of the Pen m 2 gene was then amplified by PCR, and the amplified product analyzed by electrophoresis and subcloned into the pGEM-T vector, then transformed into Escherichia coli strain JM109.…”

“…Crude extracts were analyzed by 2-D immunoblotting as described previously (26). Briefly, for the first separation, 0.5 mg of P. monodon extract was applied to an immobilized pH gradient gel strip containing pH range of 3-10 ampholytes, and isoelectric focusing was performed in a Multiphor II horizontal electrophoresis system (Amersham Pharmacia Biotech).…”