Strickler M P, Neill R J, Stone M J, Hunt R E, Brinkley W, Gemski P
Waters Chromatography Division, Millipore Corporation, Fairfax, Virginia 22030.
J Clin Microbiol. 1989 May;27(5):1031-5. doi: 10.1128/jcm.27.5.1031-1035.1989.
The Staphylococcus aureus enterotoxins represent a group of proteins that cause emesis and diarrhea in humans and other primates. We have developed a rapid two-step high-pressure liquid chromatography (HPLC) procedure for purification of staphylococcal enterotoxin B (SEB). Sterile filtrates (2.5 liters) of strain 10-275 were adsorbed directly onto a reversed-phase column (50 mm by 30 cm Delta Pak; 300 A [30 nm], 15 microns, C18). SEB was obtained by using a unique sequential gradient system. First, an aqueous ammonium acetate to acetonitrile gradient followed by an aqueous trifluoroacetic acid (TFA) wash was used to remove contaminants. A subsequent TFA to acetonitrile-TFA gradient eluted the bound SEB. Further purification was obtained by rechromatography on a cation-exchange column. From 35 to 45% of the SEB in starting filtrates was recovered. Analysis by immunoblotting of samples separated on sodium dodecyl sulfate-polyacrylamide gels indicated that HPLC-purified SEB exhibited immunological and biochemical properties similar to those of the SEB standard. Induction of an emetic response in rhesus monkeys showed that the HPLC-purified toxin also retained biological activity.
金黄色葡萄球菌肠毒素是一组可导致人类和其他灵长类动物呕吐和腹泻的蛋白质。我们开发了一种快速的两步高压液相色谱（HPLC）程序来纯化葡萄球菌肠毒素B（SEB）。将10 - 275菌株的无菌滤液（2.5升）直接吸附到反相柱（50毫米×30厘米Delta Pak；300 Å [30纳米]，15微米，C18）上。通过使用独特的连续梯度系统获得SEB。首先，使用醋酸铵水溶液到乙腈的梯度，随后用三氟乙酸（TFA）水溶液洗涤以去除污染物。随后的TFA到乙腈 - TFA梯度洗脱结合的SEB。通过在阳离子交换柱上重新色谱进一步纯化。起始滤液中35%至45%的SEB被回收。对在十二烷基硫酸钠 - 聚丙烯酰胺凝胶上分离的样品进行免疫印迹分析表明，HPLC纯化的SEB表现出与SEB标准品相似的免疫学和生化特性。在恒河猴中诱导催吐反应表明，HPLC纯化的毒素也保留了生物活性。
