PRODUCTION OF RECOMBINANT DER FI WITH THE NATIVE IGE-BINDING ACTIVITYUSING A BACULOVIRUS EXPRESSION SYSTEM

Der fI is a cysteine protease contained in feces of mites and is one o f major mite allergens, Recombinant Der fI (reDer fI) that is produced using a baculovirus expression system contains pro-sequences of diffe rent lengths. Most of these can be removed by acid treatment, However, IgE-binding activity of acid-treated reDer fI is lower than that of n ative Der fI at high protein concentrations, and N-terminal amino acid s of acid-treated reDer fI are not uniform, Now, a method for processi ng of the pro-sequence has been developed by producing reDer fI E(-1)K with baculovirus expression system in which the carboxy terminal amin o acid of the pro-sequence (glutamate) was replaced by lysine using si te directed mutagenesis, No difference in the amount of production was observed upon introducing the mutation into the pro-sequence, additio n of lysylendopeptidase into the culture medium led to processing of t he pro-sequence of reDer fI E(-1)K and proceeded the degradation of th e other proteins in the medium, Lysylendopeptidase-treated reDer fI E( -1)K was easily purified with an anion exchange column, resulting in 2 0% increase of the yield, Lysylendopeptidase-treated reDer fI E(-1)K o btained through these processes was compared with the native Der fI, A lthough some differences were found in protease activity and reactivit y with lectins, their N-terminal amino acid and the IgE-binding activi ty were the same as those of the native one, indicating its usefulness fur diagnostic purpose.