Truan Daphné, Vasil Adriana, Stonehouse Martin, Vasil Michael L, Pohl Ehmke
Swiss Light Source, Paul Scherrer Institute, 5232 Villigen, Switzerland.
Protein Expr Purif. 2013 Jul;90(1):40-6. doi: 10.1016/j.pep.2012.11.005. Epub 2012 Nov 29.
The hemolytic phospholipase C/sphingomyelinase PlcH from the opportunistic pathogen Pseudomonas aeruginosa represents the founding member of a growing family of virulence factors identified in a wide range of bacterial and fungal pathogens. In P. aeruginosa PlcH is co-expressed with a 17 kDa chaperone (PlcR2) and secreted as a fully folded heterodimer (PlcHR2) of approximately 95 kDa, by the twin arginine translocase (TAT) via the cytoplasmic membrane and through the outer membrane, by the Xcp (TypeII) secretory system. PlcHR2 has been shown to be an important virulence factor in model P. aeruginosa infections and is selectively cytotoxic, at picomolar concentrations to mammalian endothelial cells. Here we report how the various challenges starting from protein overexpression in the native organism P. aeruginosa, the use of detergents in the crystallization and data collection using the most advanced μ-focus synchrotron beam lines were overcome. Native diffraction data of this heterodimeric protein complex were collected up to a resolution of 4Å, whereas needle-shaped crystals of l-selenomethionine substituted PlcHR2 with a maximum diameter of 10 micron were used to collect data sets with a maximum resolution of 2.75Å.
来自机会致病菌铜绿假单胞菌的溶血磷脂酶C/鞘磷脂酶PlcH，是在多种细菌和真菌病原体中发现的越来越多的毒力因子家族的创始成员。在铜绿假单胞菌中，PlcH与一种17 kDa的伴侣蛋白（PlcR2）共表达，并作为一种约95 kDa的完全折叠异二聚体（PlcHR2）分泌，通过双精氨酸转运酶（TAT）经细胞质膜并通过外膜，由Xcp（II型）分泌系统分泌。PlcHR2已被证明是铜绿假单胞菌感染模型中的一种重要毒力因子，在皮摩尔浓度下对哺乳动物内皮细胞具有选择性细胞毒性。在这里，我们报告了如何克服从铜绿假单胞菌天然生物体中的蛋白质过表达开始的各种挑战，在结晶过程中使用去污剂以及使用最先进的微聚焦同步加速器光束线进行数据收集。该异二聚体蛋白复合物的天然衍射数据收集到了4Å的分辨率，而最大直径为10微米的L-硒代甲硫氨酸取代的PlcHR2针状晶体被用于收集最大分辨率为2.75Å的数据集。
