Production of recombinant Der fI with the native IgE-binding activity using a baculovirus expression system.
Der fI is a cysteine protease contained in feces of mites and is one of major mite allergens. Recombinant Der fI (reDer fI) that is produced using a baculovirus expression system contains pro-sequences of different lengths. Most of these can be removed by acid treatment. However, IgE-binding activity of acid-treated reDer fI is lower than that of native Der fI at high protein concentrations, and N-terminal amino acids of acid-treated reDer fI are not uniform. Now, a method for processing of the pro-sequence has been developed by producing reDer fI E(-1)K with baculovirus expression system in which the carboxy terminal amino acid of the pro-sequence (glutamate) was replaced by lysine using site directed mutagenesis. No difference in the amount of production was observed upon introducing the mutation into the pro-sequence. Addition of lysylendopeptidase into the culture medium led to processing of the pro-sequence of reDer fI E(-1)K and proceeded the degradation of the other proteins in the medium. Lysylendopeptidase-treated reDer fI E(-1)K was easily purified with an anion exchange column, resulting in 20% increase of the yield. Lysylendopeptidase-treated reDer fI E(-1)K obtained through these processes was compared with the native Der fI. Although some differences were found in protease activity and reactivity with lectins, their N-terminal amino acid and the IgE-binding activity were the same as those of the native one, indicating its usefulness for diagnostic purpose.
Shoji
H
H
Institute for Production Research and Development, Nikka Whisky Distilling Co., Ltd., Chiba, Japan.
Shibuya
I
I
Hirai
M
M
Horiuchi
H
H
Takagi
M
M
D000818:Animals; D000941:Antigens / Q000737:chemistry / Q000235:genetics; D039741:Antigens, Dermatophagoides; D016367:Baculoviridae; D001667:Binding, Competitive; D015153:Blotting, Western; D003470:Culture Media; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D007073:Immunoglobulin E / Q000378:metabolism; D037102:Lectins / Q000737:chemistry / Q000378:metabolism; D008925:Mites; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012697:Serine Endopeptidases / Q000737:chemistry
D000941:Antigens; D039741:Antigens, Dermatophagoides; D003470:Culture Media; D018076:DNA, Complementary; D006023:Glycoproteins; D037102:Lectins; D011994:Recombinant Proteins; D007073:Immunoglobulin E; D012697:Serine Endopeptidases; C036813:lysyl endopeptidase; D003546:Cysteine Endopeptidases