Pen c 1, a novel enzymic allergen protein from Penicillium citrinum - Purification, characterization, cloning and expression

A 33-kDa alkaline serine protease secreted by Penicillium citrinum strain 5 2-5 is shown to be an allergenic agent in this fungus. The protein, designa ted Pen c 1, was purified by sequential DEAE-Sepharose and carboxymethyl (C M)Sepharose chromatographies, Pen c 1 has a molecular mass of 33 kDa and a pi of 7.1, The caseinolytic enzyme activity of this protein was studied. Th e protein binds to serum IgE from patients allergic to Penicillium citrinum . The cDNA encoding Pen c 1 is 1420 bp in length and contains an open readi ng frame for a 397-amino-acid polypeptide. Pen c 1 codes for a larger precu rsor containing a signal peptide, a propeptide and the 33-kDa mature protei n. Sequence comparison revealed that Pen c 1 possesses several features in common with the alkaline serine proteases of the subtilisin family. The ess ential Asp, His, and Ser residues that make up the catalytic triad of serin e proteases are well conserved. Northern blots demonstrated that mRNAs tran scribed from this gene are present at early stages of culture. The allergen encoded by Pen c 1 gene was expressed in Escherichia coli as a fusion prot ein bearing an N-terminal histidine-affinity tag. The protein, purified by affinity chromatography with a yield of 130 mg of pure protein per liter of culture, was able to bind to both a monoclonal anti-Pen c 1 antibody and I gE from the serum of patients allergic to Penicillium. Recombinant Pen c 1 can therefore be expressed in E. coli in large quantities and should prove useful as a standardized specific allergen for immune-diagnosis of atopic d isorders. In addition, full caseinolytic enzyme activity could be generated in the purified recombinant protein by sulfonation and renaturation, follo wed by removal of the affinity tag, indicating that the refolded protein ca n assume the same conformation as the native protein.