Meador J, Tweten R K
Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City 73190.
Infect Immun. 1988 Jul;56(7):1708-14. doi: 10.1128/iai.56.7.1708-1714.1988.
Toxin B from Clostridium difficile was purified to homogeneity and characterized. Purification of toxin B was achieved by gel filtration, chromatography on two consecutive anion-exchange columns, and chromatography on a high-resolution anion-exchange column in the presence of 50 mM CaCl2. The molecular weight of toxin B was estimated to be 250,000 by denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 500,000 by gel filtration. No subunits were apparent when the toxin was reduced and analyzed by SDS-PAGE. The estimated molecular weight of native toxin B indicated that dimers may form in solution. Toxin B was homogeneous by SDS-PAGE, native PAGE, and high-resolution anion-exchange chromatography. No secondary sequences were detected when the amino terminus of the toxin was sequenced, which also indicated that contaminating peptides were absent from the preparation. The amino terminus of toxin B was determined to be NH3-Trp-Leu-Val-Asn-Arg-Lys-Gln-Leu-Glu-Lys-Met-Ala-Asn-Val-ARg-Phe-Arg. One cytotoxic unit of toxin B was estimated to be 0.2 to 0.8 pg.
艰难梭菌毒素B被纯化至同质并进行了特性鉴定。毒素B的纯化通过凝胶过滤、在两根连续的阴离子交换柱上进行层析以及在50 mM氯化钙存在下于高分辨率阴离子交换柱上进行层析来实现。通过变性十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）估计毒素B的分子量为250,000，通过凝胶过滤估计为500,000。当毒素经还原并用SDS-PAGE分析时，未发现亚基。天然毒素B的估计分子量表明其在溶液中可能形成二聚体。毒素B通过SDS-PAGE、天然PAGE和高分辨率阴离子交换层析呈现同质。对毒素的氨基末端进行测序时未检测到二级序列，这也表明制备物中不存在污染肽段。毒素B的氨基末端确定为NH3-Trp-Leu-Val-Asn-Arg-Lys-Gln-Leu-Glu-Lys-Met-Ala-Asn-Val-ARg-Phe-Arg。毒素B的一个细胞毒性单位估计为0.2至0.8皮克。
