Yoshimura F, Takahashi Y, Hibi E, Takasawa T, Kato H, Dickinson D P
Department of Microbiology, School of Dentistry, Aichi-Gakuin University, Nagoya, Japan.
Infect Immun. 1993 Dec;61(12):5181-9. doi: 10.1128/iai.61.12.5181-5189.1993.
Flanking DNA regions of the fimbrilin gene (designated fimA), which encodes the major subunit protein of Porphyromonas (Bacteroides) gingivalis fimbriae, were cloned in several manners from the P. gingivalis chromosome into Escherichia coli by screening with probes derived from a 2.5-kb SacI DNA fragment previously cloned. A total of 10.4 kb of DNA fragments from the P. gingivalis genome was cloned in the pUC plasmid. Expression of the fimA gene and possible flanking genes in the fragments cloned was examined in a pUC plasmid vector system and in a bacteriophage T7 RNA polymerase-promoter expression vector system. The results show that in the pUC plasmid system, a 45-kDa protein, a product of fimA, was only poorly expressed as a precursor of the fimbrilin protein (FimA) and could be detected from cell extracts in Western blotting (immunoblotting) analysis as a sharp band but not in colony immunoblotting analysis. On the other hand, in the T7 RNA polymerase-promoter system, the product of fimA and products of the possible flanking genes responsible for fimbriation were overproduced as thick bands of the 45-kDa protein and as 63-, 50-, and 80-kDa proteins, respectively, in stained electrophoresis gels. All of the recombinant proteins were insoluble and seemed to be expressed as precursors with leader peptides. The 63-kDa, 45-kDa (a truncated protein of the 50-kDa protein), and 80-kDa proteins were purified after solubilization with sodium dodecyl sulfate. N-terminal amino acid sequences of the 45-kDa and 80-kDa proteins were analyzed up to the first 35 residues with a gas-phase sequencer. Monospecific antibodies directed to the recombinant proteins, i.e., the 63-kDa, 45-k*Da, and 80-kDa proteins, were raised in rabbits. By using the antibodies, localization of their matured proteins in P. gingivalis was investigated by Western blotting analysis. Immunoblotting analysis suggests that at least the 50- and 80-kDa proteins, encoded by genes downstream from the fimA gene, are minor components associated with fimbriae.
编码牙龈卟啉单胞菌（拟杆菌属）菌毛主要亚基蛋白的菌毛蛋白基因（命名为fimA）的侧翼DNA区域，通过用先前克隆的2.5kb SacI DNA片段衍生的探针进行筛选，以多种方式从牙龈卟啉单胞菌染色体克隆到大肠杆菌中。总共10.4kb的牙龈卟啉单胞菌基因组DNA片段被克隆到pUC质粒中。在pUC质粒载体系统和噬菌体T7 RNA聚合酶-启动子表达载体系统中检测克隆片段中fimA基因及可能的侧翼基因的表达。结果表明，在pUC质粒系统中，菌毛蛋白的前体（FimA）作为fimA的产物的一种45kDa蛋白表达不佳，并且在蛋白质印迹（免疫印迹）分析中可以从细胞提取物中检测到清晰条带，但在菌落免疫印迹分析中未检测到。另一方面，在T7 RNA聚合酶-启动子系统中，fimA的产物以及负责菌毛形成的可能侧翼基因的产物在染色电泳凝胶中分别作为45kDa蛋白的浓条带以及63kDa、50kDa和80kDa蛋白过量产生。所有重组蛋白均不溶，似乎以前体形式与前导肽一起表达。用十二烷基硫酸钠溶解后，纯化了63kDa、45kDa（50kDa蛋白的截短蛋白）和80kDa蛋白。用气相测序仪分析了45kDa和80kDa蛋白的N端氨基酸序列，直至前35个残基。针对重组蛋白（即63kDa、45k*Da和80kDa蛋白）制备了单特异性抗体。通过使用这些抗体，通过蛋白质印迹分析研究了它们的成熟蛋白在牙龈卟啉单胞菌中的定位。免疫印迹分析表明，至少由fimA基因下游基因编码的50kDa和80kDa蛋白是与菌毛相关的次要成分。
