Fredriksen J H, Frøholm L O, Paulsen B S
Dev Biol Stand. 1985;61:187-96.
One serotype antigen, agglutinogen 3, from Bordetella pertussis, (strain M2, serotype 1.3), has been purified. The purification procedure included acetone drying of cells harvested from shaking cultures. Agglutinogens were extracted in phosphate buffered saline. Crude extract was heat treated at 80 degrees C for 5 min and precipitated by ammonium sulphate between 25 and 60% saturation at 4 degrees C, providing 50% of the total activity and a five-fold purification. Further purification was attempted by gel filtration chromatography using a TSK-G3000 SW column. The ammonium sulphate precipitated fraction was also separated by anion exchange chromatography using a Mono Q HR 5/5 column. The purification work indicated that agglutinogen 3 is associated with several other substances and that this property can lead to purification difficulties. The isolation procedure was monitored by an agglutination-inhibition assay. The peak fraction from the ion exchange chromatography was purified up to 27-fold according to the specific activities (inhibition units per mg protein). The yield was only 1% due to severe loss of activity. In the gel filtration chromatography agglutinogen 3-activity eluted with a maximum activity corresponding to a molecular weight near 450,000. SDS-PAGE analysis indicated that agglutinogen 3 might have a subunit molecular weight of 20,800.
来自百日咳博德特氏菌（菌株M2，血清型1.3）的一种血清型抗原——凝集原3已被纯化。纯化过程包括对从振荡培养物中收获的细胞进行丙酮干燥。凝集原在磷酸盐缓冲盐溶液中提取。粗提取物在80℃热处理5分钟，然后在4℃下用饱和度为25%至60%的硫酸铵沉淀，得到了50%的总活性和5倍的纯化效果。尝试使用TSK - G3000 SW柱通过凝胶过滤色谱法进一步纯化。硫酸铵沉淀部分也使用Mono Q HR 5/5柱通过阴离子交换色谱法进行分离。纯化工作表明凝集原3与其他几种物质相关，并且这种特性可能导致纯化困难。分离过程通过凝集抑制试验进行监测。根据比活性（每毫克蛋白质的抑制单位），离子交换色谱法的峰值部分纯化至27倍。由于活性严重损失，产率仅为1%。在凝胶过滤色谱法中，凝集原3活性以最大活性洗脱，对应分子量接近450,000。SDS - PAGE分析表明凝集原3可能具有20,800的亚基分子量。
