Production of Recombinant Der fI with the Native IgE-Binding Activity Using a Baculovirus Expression System

Abstract

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.

Production of Enzymatically and Immunologically Active Der f 1 in <i>Escherichia coli</i>

“…To overcome the poor availability of purified allergen, the production of recombinant group 1 allergen has been attempted. Recombinant Der f 1 (rDer f 1) was successfully produced in a Baculovirus expression system [9, 10]. However, in addition to the high costs of the medium, the product had a sugar chain different from the native one.…”

A Recombinant Group 1 House Dust Mite Allergen, rDer f 1, with Biological Activities Similar to Those of the Native Allergen

“…To date, the most promising system for producing a rDer f 1 is a baculovirus expression system developed by Shoji and coworkers (11,12). These investigators constructed a recombinant baculovirus that included the baculovirus (Autographica californica nuclear polyhedrosis) polyhedrin promoter and a region encoding the 18 residue Der f 1 signal peptide, the 80 residue pro-sequence, and the entire mature Der f 1.…”

“…The IgE binding activity of the pro-form could be enhanced to a level comparable to the native antigen if the pro-peptide was removed in a postpurification protease digestion step. Although Shoji and coworkers' work (11,12) is encouraging, their process and assay results have some limitations: (1) Der f 1 contains internal lysine residues. A nonspecific protease such as lysylendopeptidase cannot be easily controlled to cleave only at the engineered lysine outside the mature protein and not at any of the internal lysines in the mature protein.…”

“…In addition, use of a protease adds additional steps and costs to the process. (2) Shoji and coworkers' IgE binding studies used sera pooled from a collection of human patients (11,12). Patients with low levels of antigen-specific IgE may be under represented or omitted from pooled sera.…”

Effects of Site-Directed Mutagenesis in the Cysteine Residues and the N-Glycosylation Motif in Recombinant Der f 1 on Secretion and Protease Activity

“…Native Der f 1 is glycosylated [31]. rDer f 1 molecules expressed in yeast and insect cells are considered to have the glycochain whose structure is different from that of native Der f 1.…”