Sojar H T, Hamada N, Genco R J
Department of Oral Biology, School of Dental Medicine, State University of New York at Buffalo, 14214, USA.
Protein Expr Purif. 1997 Feb;9(1):49-52. doi: 10.1006/prep.1996.0662.
Fimbriae are responsible for the adherence of many bacterial species to the surfaces they eventually colonize. Porphyromonas gingivalis is an important pathogenic agent involved in periodontal disease. Fimbriae of P. gingivalis have been thought to mediate binding of the bacterium to oral surfaces. In order to study the role of fimbriae in microbial adhesion, it is important to purify fimbriae to homogeneity. A simple and rapid reverse-phase high-performance liquid chromatography (HPLC) method is developed to purify P. gingivalis fimbriae. The crude fimbriae were precipitated from sonic extract of P. gingivalis cells with the 40% ammonium sulfate precipitation. The dialyzed crude fimbriae preparation was subjected to reverse-phase HPLC separation. The purity and size of purified fimbrial proteins were confirmed by SDS-polyacrylamide gel electrophoresis(SDS-PAGE) followed by Western immunoblot using polyclonal antibodies against fimbriae. The purified major fimbrial protein from strain 33277 of P. gingivalis had apparent molecular mass of 41 kDa. The method is useful for analytical as well as preparative purification with 25% yields from the ammonium sulfate-precipitated crude fimbriae preparation, and represents increased speed and efficiency over earlier published methods.
菌毛负责许多细菌物种黏附到它们最终定植的表面。牙龈卟啉单胞菌是参与牙周疾病的一种重要病原体。牙龈卟啉单胞菌的菌毛被认为介导该细菌与口腔表面的结合。为了研究菌毛在微生物黏附中的作用，将菌毛纯化至同质状态很重要。开发了一种简单快速的反相高效液相色谱（HPLC）方法来纯化牙龈卟啉单胞菌菌毛。用40%硫酸铵沉淀法从牙龈卟啉单胞菌细胞的超声提取物中沉淀粗菌毛。将透析后的粗菌毛制剂进行反相HPLC分离。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE），随后使用针对菌毛的多克隆抗体进行Western免疫印迹，来确认纯化菌毛蛋白的纯度和大小。从牙龈卟啉单胞菌33277菌株纯化的主要菌毛蛋白的表观分子量为41 kDa。该方法对于分析纯化和制备纯化均有用，从硫酸铵沉淀的粗菌毛制剂中获得25%的产率，并且与早期发表的方法相比，速度更快、效率更高。
