Purification of Staphylococcal Alpha-Toxin

The strain most widely used tor toxin production is S. aureus Wood 46. Note that the presence of proteases may give rise to proteolytic cleavage of alpha-toxin, which results in altered pore-forming activity (Palmer et al., 1993). Moreover, proteolytic activity contaminating the final toxin preparation may interfere with cell-biological experiments. Per liter of culture, the following protocol typically yields 20 mg of alpha-toxin suitable for cell-biological applications with a strain available from this laboratory. 

The protocol given below requires a suitable membrane filtration device (molecular weight cut-off below 30kDa effective surface ca. 0.5 m for 2-31 of culture) as available from Filtron or Millipore for concentration of culture supernatants. 

2 X TY medium dissolve 20g/l Bacto-Tryptone (Difco), 10 g yeast extract (Difco), and 10 g NaCI in 1 I water, and autoclave 

Column buffer A ammonium acetate 20 mM, pH 6.2 Column buffer B ammonium acetate 500 mM, pH 6.2 

2xTY medium is used. A starter culture of 30 ml is inoculated, shaken overnight and diluted 1 100 with 2xTY to yield the production culture. To achieve good aeration, the production culture is divided into portions of 500 ml contained in 21 Erlenmeyer flasks, which are incubated in an orbitary shaker tor 18 h (37°C, 250 r.p.m in an orbitary shaker (New Brunswick Scientific G25). 

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