Production and applications of recombinant proteinase 3, Wegener's autoantigen: problems and perspectives.
Antineutrophil cytoplasmic antibodies (cANCA) against conformational epitopes ofproteinase 3 (PR3) are regarded as an important pathogenic marker in Wegener's granulomatosis (WG). Hence, PR3-based antibody binding assays are widely used for diagnosis and monitoring of the disease. Purification of the native catalytically active serine protease from granulocytes, however, is relatively inefficient, time-consuming and technically demanding. Conformational changes, partial aggregation, denaturation during purification and contaminations with inhibitors or other proteins from plasma and granulocytes, can affect the quality and comparability of PR3-based cANCA determinations. Alternative production of the human PR3 autoantigen by recombinant technologies offers several advantages over the natural antigen, but the complexity and operating expense of these procedures have, so far, delayed the development of new clinical tests. Correct posttranslational processing, conformational identity and antigen stability can be achieved by the expression of PR3 in Sf9 insect cells and in mammalian hosts, HEK293 and HMC-1. Subsequent purification and immobilization of the recombinant antigen is furthermore simplified by the attachment of short carboxy-terminal peptide tags. In contrast to conventional capture techniques with murine monoclonal antibodies, tag-based immobilization of the recombinant antigen does not mask portions of the PR3 surface and improves the efficacy of antigen coating. Moreover, recombinant PR3 variants will have a great potential to study individual anti-PR3 responses and will advance the development of new epitope-based therapeutics.
Jenne
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Kuhl
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D000595:Amino Acid Sequence; D001324:Autoantigens / Q000096:biosynthesis* / Q000737:chemistry; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D053146:Myeloblastin / Q000096:biosynthesis* / Q000737:chemistry; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry
D001324:Autoantigens; D011994:Recombinant Proteins; D053146:Myeloblastin