Satoshi Koyanagi

A convenient method for preparation of the calcium ion-binding protein annexin V

This study presents a novel method for the production and purification of annexin V using cation exchange chromatography in the presence of calcium ions at neutral or alkaline pH. This method enables the handling of a large quantity of sample at one time without deterioration of the adsorption capacity of the cation exchange carrier by contaminating proteins, as well as the production of the calcium ion-binding protein annexin V with high purity on an industrial scale from both recombinant products and native products.

Large-scale production of major house dust mite allergen Der f 2 mutant (C8/119S) in Escherichia coli

Hyposensitization, in which causative antigens of allergic diseases are injected, is the sole means of a radical cure for allergic diseases. Since the therapeutic allergens currently used are naturally extracted, producing preparations with a stable titer from such extracts is extremely difficult. There are several reports on the expression of recombinant mite allergens in Escherichia coli using inducers. The use of an inducer for industrial production will lead to high costs and, for therapeutic use, it must be removed in the purification process. C8/119S is a mutant of Der f 2, a major house dust mite allergen. The C8/119S gene was integrated downstream of the trp promoter to produce the expression plasmid (pWU11-C8/119S). Then, this expression plasmid was used to transform E. coli strain HB101 (pWU11-C8/119S/HB101). A recombinant E. coli pWU11-C8/119S/HB101 did not express C8/119S in a low-temperature culture (32 degrees C), but C8/119S was induced to a high level of expression in a high-temperature culture (37 degrees C). pWU11-C8/119S/HB101 proliferated when expression was induced by high temperature and an approximately 3-fold greater proliferation was obtained compared with the use of an inducer in a large-scale culture. The C8/119S protein was expressed as inclusion bodies and obtained by refolding and chromatography purifications. The immunological properties of C8/119S were assessed by western blotting. Western blotting demonstrated that purified C8/119S reacted with a monoclonal anti-Der f 2 antibody (18G8). pWU11-C8/119S/HB101 can be used as an easy, low cost expression system on a large scale. It is also advantageous for industrial production in that the addition of an inducer is not required to achieve expression of the mite allergen.

Production-scale purification of the recombinant major house dust mite allergen Der f 2 mutant C8/119S.

A WHO position paper states that allergen immunotherapy is an effective treatment for allergic diseases, and well characterized allergens should be used in immunotherapy. The house dust mite is a major cause of allergic disease. However, the biological activity of the mite extracts currently used cannot be clearly determined, since these extracts contain various impurities. The use of recombinant allergens can avoid this problem. However, there remains a risk of contamination by other impurities, such as host cell-derived proteins (HCPs). Advanced purification techniques are thus required to remove these contaminants. C8/119S is a mutant of the major house dust mite allergen Der f 2, and is expressed and accumulated as an inclusion body in Escherichia coli. The C8/119S was refolded and purified through three column chromatography steps. Using this method, we could obtain about 2g of the purified C8/119S in one purification batch. This amount is equivalent to 100,000 of the maintenance doses required for immunotherapy based on the WHO position paper. The purity of the C8/119S was 99% or more. The antigenicity of HCPs in the C8/119S was examined by passive cutaneous anaphylaxis assays. When the C8/119S was administered at 40 μg/kg, no local anaphylaxis was observed. C8/119S was thus highly purified with an extremely low level of impurities, and our procedure was shown to be an effective advanced production-scale purification process for this Der f 2 mutant. In this study, we established an advanced purification processes for C8/119S, then characterized the purified C8/119S and evaluated its purity.

Characterization of the Recombinant Der F 2 Mutant C8/119S and Evaluation of C8/119S in a Rder F 2-Sensitized Rhinitis Mice Model

Immunotherapy is the only curative approach to treat allergy, but carries the risk of anaphylaxis. C8/119S is a mutant of Der f 2, which is one of the causative allergens of perennial allergic diseases, and has been selected to decrease the risk of anaphylaxis in immunotherapy. In this study, the physical properties of C8/119S were determined, and the efficacy of C8/119S was evaluated in a NC/Nga mouse rhinitis model. C8/119S and recombinant Der f 2 (rDer f 2) were expressed in Escherichia coli . Purified allergens were analyzed by physicochemical and immunological techniques. In addition, rhinitis was provoked in rDer f 2-sensitized NC/Nga mice by nasal administration of rDer f 2, and C8/119S was administered. After provocation tests with rDer f 2, the number of eosinophils infiltrating the nasal mucosa was determined. C8/119S had a disordered structure, and the binding activity of allergic patients’ IgE to C8/119S was decreased compared with rDer f 2. In the NC/Nga mouse rhinitis model, eosinophil infiltration provoked by rDer f 2 was significantly controlled by the administration of C8/119S. Although similar therapeutic effects were also observed with rDer f 2 administration, 11 of 20 animals died during the rDer f 2 treatment period. On the other hand, no deaths occurred during C8/119S treatment. C8/119S appears to be an effective allergen vaccine for immunotherapy in patients with mite allergy and also appears to be safer than wild-type allergen vaccines.