Production of the bullous pemphigoid antigen 230 (BP230) by Saccharomyces cerevisiae and Pichia pastoris

Article Article scientifique Article Production of the bullous pemphigoid antigen 230 (BP230) by Saccharomyces cerevisiae and Pichia pastoris eng 2003-06 author Laffitte Emmanuel Alexis Department of Dermatology, University Hospital, Rue Micheli-du-Crest 24, CH-1211 Geneva 4, Switzerland. 520456 Département de médecine (DEMED) author Shafaatian Saied Mohammad Reza 37809 author Fontao Lionel 514347 author Favre Bertrand author Koster Jan author Saurat Jean-Hilaire 21995 Décanat et administration de la faculté de médecine (ADMED) author Monod Michel author Borradori Luca 120287 141-147 10.1016/s1046-5928(03)00057-3 12767802 1046-5928 https://www.sciencedirect.com/science/article/pii/S1046592803000573 BP230 is a cytoskeletal linker protein of 2649 amino acids originally identified as the target autoantigen in bullous pemphigoid, a potentially devastating autoimmune skin blistering disorder. To better define its function, we sought to generate recombinant forms of BP230 in both Saccharomyces cerevisiae and Pichia pastoris after cloning its entire cDNA. By immunoblot analysis, full-length BP230 was not found in extracts of P. pastoris, whereas minor amounts of degraded BP230 were detected in extracts of S. cerevisiae. In contrast, both S. cerevisiae and P. pastoris were able to produce the 770-amino acid COOH-terminal domain of BP230. Furthermore, the production level of the recombinant BP230 tail in S. cerevisiae was significantly higher than that observed in P. pastoris and that of endogenous BP230 in cultured human keratinocytes. Finally, 12 of 17 (71%) BP sera recognized the recombinant BP230 protein in yeast extracts. Our results indicate that S. cerevisiae occasionally constitutes a better tool for recombinant protein production than P. pastoris. Although both its large size and poor solubility limit production of BP230, the developed yeast system provides cellular fractions enriched in BP230 recombinant proteins that constitute useful tools for the diagnosis of bullous pemphigoid. Antigens, Fungal / biosynthesis Antigens, Fungal / chemistry Antigens, Fungal / genetics Autoantigens / biosynthesis Autoantigens / chemistry Autoantigens / genetics Carrier Proteins Cells, Cultured Collagen / biosynthesis Collagen / chemistry Collagen / genetics Cytoskeletal Proteins Dystonin Humans Immunoblotting Keratinocytes / metabolism Nerve Tissue Proteins Non-Fibrillar Collagens Pemphigoid, Bullous / blood Pichia / immunology Protein Structure, Tertiary Recombinant Proteins / genetics Recombinant Proteins / metabolism Saccharomyces cerevisiae / immunology Solubility Dewey 616 56 Département de médecine 221 false Protein expression and purification 29 2 9a182c1d-5381-4138-a8ae-a9ccb927839e Article (Published version) Restricted 1-s2.0-S1046592803000573-main.pdf 229026 application/pdf 763ed49743245d3b31259fe349e9b4a52a7665d96ffd9ecbd79607829afdc66f restrictedAccess Faculté de médecine / Section de médecine clinique / Département de médecine

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Production of the bullous pemphigoid antigen 230 (BP230) by Saccharomyces cerevisiae and Pichia pastoris	Protein expression and purification			2003	85	0

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Shafaatian, Saied Mohammad Reza