Foster A J, Bird R A, Smith S N
Life and Health Sciences, Aston University, Birmingham, UK.
J Appl Microbiol. 2007 Aug;103(2):390-9. doi: 10.1111/j.1365-2672.2006.03259.x.
To develop a novel procedure for isolating and characterizing cryptococcal cell-surface proteins using biotinylation, fluorescein isothiocyanate (FITC)-streptavidin, flow cytometry and associated ligand-receptor analysis, confocal microscopy and electrophoretic separation.
Cell proteins of both acapsulate and encapsulated Cryptococcus neoformans cells were labelled using sulfo-NHS-biotin which, in turn, was complexed with FITC-streptavidin. Resulting cell population fluorescence supported visualization of cell-surface protein distribution by confocal microscopy, as well as evaluation of protein exposure by flow cytometry and the calculation of the ligand-binding determinants EC(50), F(max) and H(n). Biotinylation of cell-surface proteins also supported their isolation by affinity chromatography and characterization by SDS/PAGE. Ligand-binding determinants, such as EC(50) values, indicated that acapsulate and stationary phase cells have greatest affinity for biotin. F(max) values demonstrated greatest protein exposure among stationary phase cells; in turn, encapsulated cells expose more protein than acapsulate counterparts. H(n) values of below unity potentially confirm the complex multi-receptor nature of biotin binding to cryptococcal cell surfaces under investigation. Fluorescence visualization showed marked but localized fluorescence indicative of protein exposure around sites of cell division. In turn, biotinylation of cell-surface proteins and their release under reducing conditions demonstrated at least two noncovalently linked proteinaceous entities, of 43 and 57 kDa, exposed on acapsulate cryptococcal cell walls.
A novel method for identifying, in situ, cell-surface proteins exposed by C. neoformans was established. This novel technique was successfully implemented using both acapsulate and encapsulated C. neoformans cells, both were found to have dynamic and markedly localized protein distribution around sites of cell division and associated cell wall trauma.
A novel procedure, employing a versatile combination of flow cytometry, ligand-receptor analysis, confocal microscopy and biotinylation, supported the characterization and isolation of cryptococcal cell-surface proteins.
开发一种使用生物素化、异硫氰酸荧光素（FITC）-链霉亲和素、流式细胞术及相关配体-受体分析、共聚焦显微镜和电泳分离来分离和鉴定隐球菌细胞表面蛋白的新方法。
使用磺基-NHS-生物素标记无荚膜和有荚膜新型隐球菌细胞的细胞蛋白，磺基-NHS-生物素再与FITC-链霉亲和素结合。所得细胞群体荧光支持通过共聚焦显微镜观察细胞表面蛋白分布，以及通过流式细胞术评估蛋白暴露情况，并计算配体结合决定因素EC(50)、F(max)和H(n)。细胞表面蛋白的生物素化还支持通过亲和色谱法分离它们，并通过SDS/PAGE进行鉴定。配体结合决定因素，如EC(50)值，表明无荚膜和静止期细胞对生物素具有最大亲和力。F(max)值表明静止期细胞中蛋白暴露最多；反过来，有荚膜细胞比无荚膜细胞暴露更多蛋白。低于1的H(n)值可能证实了正在研究的生物素与隐球菌细胞表面结合的复杂多受体性质。荧光可视化显示出明显但局部的荧光，表明细胞分裂部位周围有蛋白暴露。反过来，细胞表面蛋白的生物素化及其在还原条件下的释放表明，无荚膜隐球菌细胞壁上暴露有至少两个非共价连接的蛋白质实体，分子量分别为43 kDa和57 kDa。
建立了一种原位鉴定新型隐球菌暴露的细胞表面蛋白的新方法。这项新技术在无荚膜和有荚膜新型隐球菌细胞中均成功实施，发现两者在细胞分裂部位和相关细胞壁损伤部位周围均具有动态且明显局部化的蛋白分布。
一种采用流式细胞术、配体-受体分析、共聚焦显微镜和生物素化的多功能组合的新方法，支持了隐球菌细胞表面蛋白的鉴定和分离。
