Journal article
Purification of Staphylococcus aureus beta-toxin: comparison of three isoelectric focusing methods
Protein expression and purification, Vol.9(1), pp.76-82
02/1997
DOI: 10.1006/prep.1996.0664
PMID: 9116505
Abstract
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.
Details
- Title: Subtitle
- Purification of Staphylococcus aureus beta-toxin: comparison of three isoelectric focusing methods
- Creators
- Dara K Gaskin - Department of Microbiology, University of Idaho, Moscow 83844, USAGregory A BohachPatrick M SchlievertCarolyn J Hovde
- Resource Type
- Journal article
- Publication Details
- Protein expression and purification, Vol.9(1), pp.76-82
- Publisher
- United States
- DOI
- 10.1006/prep.1996.0664
- PMID
- 9116505
- ISSN
- 1046-5928
- eISSN
- 1096-0279
- Grant note
- A128401 / PHS HHS A133981 / PHS HHS
- Language
- English
- Date published
- 02/1997
- Academic Unit
- Microbiology and Immunology; Internal Medicine
- Record Identifier
- 9984001207102771
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