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 was 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.
Central Research Laboratories, Asahi Breweries, Ltd., Tokyo, Japan.
C
C
Nishiyama
T
T
Yasuhara
T
T
Yuuki
Y
Y
Okumura
D000485:Allergens; D000595:Amino Acid Sequence; D039741:Antigens, Dermatophagoides; D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005982:Glutathione Transferase / Q000235:genetics; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007073:Immunoglobulin E / Q000378:metabolism; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases / Q000378:metabolism
D000485:Allergens; D039741:Antigens, Dermatophagoides; D018076:DNA, Complementary; D006023:Glycoproteins; D011993:Recombinant Fusion Proteins; D007073:Immunoglobulin E; D005982:Glutathione Transferase; D012697:Serine Endopeptidases