Gaskin D K, Bohach G A, Schlievert P M, Hovde C J
Department of Microbiology, University of Idaho, Moscow 83844, USA.
Protein Expr Purif. 1997 Feb;9(1):76-82. doi: 10.1006/prep.1996.0664.
beta-Toxin (beta-hemolysin) is one of several extracellular proteins produced by Staphylococcus aureus. It is a sphingomyelinase which disrupts the membranes of erythrocytes and other mammalian cells. Despite its characterized mechanism of action, the role of beta-toxin in human and animal disease remains unclear. In this report, we compare three gentle, rapid methods to purify enzymatically active beta-toxin. Extracellular proteins in S. aureus strain RN4220 cell supernatants, containing a high concentration of the toxin, were precipitated by ethanol, dialyzed, and separated by preparative isoelectric focusing (IEF). We compared the efficiency of three preparative IEF methods: a Sephadex flat-bed IEF using pH 3.5-10.0 Ampholine, a liquid IEF using pH 7.8-8.9 Rotolyte buffers, and a liquid IEF with two consecutive steps using pH 3.0-10.0 Bio-Lytes in the first separation followed by a second step using pH 6.0-8.0 Bio-Lytes. All three IEF methods purified milligram amounts of enzymatically and biologically active beta-toxin. Typically, 2-5 mg of purified beta-toxin was obtained from 1.2 liters of culture medium. The total enzymatic activity recovered and overall yield were similar for all three methods. However, the single-step liquid IEF separation using Rotolyte buffers was the most preferred method because it purified beta-toxin to >95% purity, did not require dialysis to remove ampholytes, and was the most rapid of the three methods.
β毒素（β溶血素）是金黄色葡萄球菌产生的几种细胞外蛋白之一。它是一种鞘磷脂酶，可破坏红细胞和其他哺乳动物细胞的膜。尽管其作用机制已得到明确，但β毒素在人类和动物疾病中的作用仍不清楚。在本报告中，我们比较了三种温和、快速的方法来纯化具有酶活性的β毒素。金黄色葡萄球菌RN4220菌株细胞上清液中的细胞外蛋白含有高浓度的毒素，通过乙醇沉淀、透析，并通过制备性等电聚焦（IEF）进行分离。我们比较了三种制备性IEF方法的效率：使用pH 3.5 - 10.0两性电解质的Sephadex平板IEF、使用pH 7.8 - 8.9 Rotolyte缓冲液的液相IEF，以及在第一次分离中使用pH 3.0 - 10.0 Bio-Lytes，第二步使用pH 6.0 - 8.0 Bio-Lytes的两步连续液相IEF。所有三种IEF方法都纯化出了毫克量的具有酶活性和生物活性的β毒素。通常，从1.2升培养基中可获得2 - 5毫克纯化的β毒素。所有三种方法回收的总酶活性和总产率相似。然而，使用Rotolyte缓冲液的单步液相IEF分离是最优选的方法，因为它将β毒素纯化至纯度>95%，不需要透析去除两性电解质，并且是三种方法中最快的。
