Cloning and expression in Escherichia coli of cDNA encoding house dust mite allergen Der f 3, serine protease from Dermatophagoides farinae

Der f 3 is one of the allergens produced by house dust mite Dermatophagoides farinae showing serine protease activity, Based on its amino acid sequence, a cDNA clone encoding Der f 3 was isolated from a cDNA library of D. farinae, Sequencing analysis of the clone revealed the presence of an open reading frame of 780 bp, which encodes a mature protein of 232 amino acids with 27 amino acids of pre-pro sequence at the N-terminus. When proDer f 3 was produced in Escherichia coli as a fused protein with glutathione-S-transferase, the fused protein was accumulated as inclusion bodies, The protein purified with 8 M urea and glutathione-affinity column chromatography, however, did not show protease activity, When an arginine residue,vas introduced at the C-terminus of the pro-region in place of threonine, removal of the pro-region to produce an active mature protease was observed, The specificity and the activity of this recombinant protease were almost the same as those of native Der f 3.