Laffitte Emmanuel, Shafaatian Reza, Fontao Lionel, Favre Bertrand, Koster Jan, Saurat Jean-Hilaire, Monod Michel, Borradori Luca
Department of Dermatology, University Hospital, Rue Micheli-du-Crest 24, CH-1211 Geneva 4, Switzerland.
Protein Expr Purif. 2003 Jun;29(2):141-7. doi: 10.1016/s1046-5928(03)00057-3.
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.
BP230是一种由2649个氨基酸组成的细胞骨架连接蛋白，最初被鉴定为大疱性类天疱疮（一种潜在的毁灭性自身免疫性皮肤水疱病）中的靶自身抗原。为了更好地确定其功能，我们在克隆了BP230的完整cDNA后，试图在酿酒酵母和毕赤酵母中产生其重组形式。通过免疫印迹分析，在毕赤酵母提取物中未发现全长BP230，而在酿酒酵母提取物中检测到少量降解的BP230。相比之下，酿酒酵母和毕赤酵母都能够产生BP230的770个氨基酸的COOH末端结构域。此外，酿酒酵母中重组BP230尾部的产生水平显著高于毕赤酵母中观察到的水平以及培养的人角质形成细胞中内源性BP230的水平。最后，17份BP血清中有12份（71%）识别酵母提取物中的重组BP230蛋白。我们的结果表明，酿酒酵母有时构成比毕赤酵母更好的重组蛋白生产工具。尽管其大尺寸和低溶解性限制了BP230的生产，但所开发的酵母系统提供了富含BP230重组蛋白的细胞组分，这些组分构成了诊断大疱性类天疱疮的有用工具。
