Masuoka J, Glee P M, Hazen K C
Department of Pathology, University of Virginia Medical Center, Charlottesville 22908, USA. jm2n@Virginia-edu
Electrophoresis. 1998 May;19(5):675-8. doi: 10.1002/elps.1150190512.
Hydrophobic proteins in the cell wall of the opportunistic fungal pathogen Candida albicans are involved in adhesion of this organism to host tissue and thus play a role in its pathogenicity. The hydrophobic nature of these proteins results in their loss during purification due to adsorption to apparatus surfaces. This problem, combined with their low abundance, has made it problematic to purify the hydrophobic proteins in sufficient quantity for sequencing or biochemical analysis. We describe a system that combines preparative isoelectric focusing with continuous elution preparative electrophoresis. The system provides a two-dimensional protein separation while maintaining protein solubility and minimizing protein loss due to adsorption. In addition, we have added an in-line transfer of electrophoretic fractions directly to polyvinylidene difluoride (PVDF) membranes, which further reduces both exposure to apparatus surfaces and purification time.
机会性真菌病原体白色念珠菌细胞壁中的疏水蛋白参与该生物体与宿主组织的黏附，因此在其致病性中发挥作用。这些蛋白质的疏水性导致它们在纯化过程中因吸附到仪器表面而损失。这个问题，再加上它们的低丰度，使得难以纯化出足够量的疏水蛋白用于测序或生化分析。我们描述了一种将制备型等电聚焦与连续洗脱制备型电泳相结合的系统。该系统提供二维蛋白质分离，同时保持蛋白质溶解性并使因吸附导致的蛋白质损失最小化。此外，我们增加了将电泳组分直接在线转移到聚偏二氟乙烯（PVDF）膜上的步骤，这进一步减少了与仪器表面的接触以及纯化时间。
