Production of the bullous pemphigoid antigen 230 (BP230) by Saccharomyces cerevisiae and Pichia pastoris.
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.
Laffitte
Emmanuel
E
Department of Dermatology, University Hospital, Rue Micheli-du-Crest 24, CH-1211 Geneva 4, Switzerland.
Shafaatian
Reza
R
Fontao
Lionel
L
Favre
Bertrand
B
Koster
Jan
J
Saurat
Jean-Hilaire
JH
Monod
Michel
M
Borradori
Luca
L
D000946:Antigens, Fungal / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D001324:Autoantigens / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D002352:Carrier Proteins; D002478:Cells, Cultured; D003094:Collagen / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D003598:Cytoskeletal Proteins; D000071821:Dystonin; D006801:Humans; D015151:Immunoblotting; D015603:Keratinocytes / Q000378:metabolism; D009419:Nerve Tissue Proteins; D024041:Non-Fibrillar Collagens; D010391:Pemphigoid, Bullous / Q000097:blood; D010843:Pichia / Q000276:immunology*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000276:immunology*; D012995:Solubility; D000097805:Collagen Type XVII
D000946:Antigens, Fungal; D001324:Autoantigens; D002352:Carrier Proteins; D003094:Collagen; D003598:Cytoskeletal Proteins; D000071821:Dystonin; D009419:Nerve Tissue Proteins; D024041:Non-Fibrillar Collagens; D011994:Recombinant Proteins; D000097805:Collagen Type XVII; C486268:DST protein, human