Lee J Y, Sojar H T, Amano A, Genco R J
Department of Oral Microbiology, School of Dentistry, Kyung Hee University, Seoul, Korea.
Protein Expr Purif. 1995 Aug;6(4):496-500. doi: 10.1006/prep.1995.1066.
Binding of Porphyromonas gingivalis to pellicle-coated teeth is mediated to a large extent by fimbrillin, a major structural subunit of fimbriae. A simple and efficient method for purifying the major fimbrial proteins from various strains of P. gingivalis was developed. The sonic extracts of crude fimbriae were prepared from P. gingivalis strains 2561, A7A1-28, EM-3, and 9-14K-1, which are representatives of different fimbrial groups. The crude fimbriae were precipitated from the extracts with 40% saturated ammonium sulfate. The dialyzed crude fimbriae were dissolved in 8 M guanidine HCl and loaded onto a Sepharose CL-6B column equilibrated with 6 M guanidine HCl. The major fimbrial protein-enriched fractions were obtained and further purified to homogeneity by repetitive gel filtration on the same column. The purity and intactness of the purified fimbrial proteins were ascertained by SDS-polyacrylamide gel electrophoresis followed by silver nitrate staining and immunoblot analysis using rabbit antisera raised against the purified fimbrial proteins. The result demonstrates the usefulness of guanidine HCl as a reliable reagent for purifying various fimbrial proteins of P. gingivalis, which are often associated strongly with other outer membrane proteins and show different physicochemical and antigenic characteristics.
牙龈卟啉单胞菌与牙面获得性膜的结合在很大程度上是由菌毛素介导的，菌毛素是菌毛的主要结构亚基。开发了一种从牙龈卟啉单胞菌不同菌株中纯化主要菌毛蛋白的简单有效方法。从2561、A7A1 - 28、EM - 3和9 - 14K - 1等牙龈卟啉单胞菌菌株制备粗菌毛的超声提取物，这些菌株代表不同的菌毛组。用40%饱和硫酸铵从提取物中沉淀粗菌毛。将透析后的粗菌毛溶解于8M盐酸胍中，加载到用6M盐酸胍平衡的琼脂糖CL - 6B柱上。获得富含主要菌毛蛋白的组分，并通过在同一柱上重复凝胶过滤进一步纯化至均一。通过SDS - 聚丙烯酰胺凝胶电泳，随后进行硝酸银染色以及使用针对纯化菌毛蛋白制备的兔抗血清进行免疫印迹分析，确定纯化菌毛蛋白的纯度和完整性。结果表明盐酸胍作为一种可靠的试剂，可用于纯化牙龈卟啉单胞菌的各种菌毛蛋白，这些菌毛蛋白通常与其他外膜蛋白紧密结合，并表现出不同的物理化学和抗原特性。
