Production, purification, and chemical characterization of Staphylococcus aureus exfoliative toxin

Abstract

Methods for the production and isolation of exfoliative toxin are described. Fermentation conditions were established under which large quantities of the crude material can be produced. Column chromatography methods, including carboxymethyl cellulose and hydroxyapatite, were utilized to purify the toxic protein. The pure toxin had a molecular weight of 26,000 as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The pure toxin is a simple protein composed of 17 amino acids. Tests for carbohydrate and for alpha- and beta-hemolysin were negative. The mean effective dose of the purified toxin was 0.5 mug per newborn mouse.

Partial purification and characterization of a heat-labile enterotoxin of Escherichia coli

“…Tryptophan was not determined. Remarkable is the total absence of half-cystine and methionine, though this has been reported previously for other exoproteins from E. coli (19) as well as more recently for the exfoliative toxin from Staphylococcus aureus (14). The absence of cysteine or cystine was confirmed by subjecting the material to exhaustive reduction and alkylation with ['4C]iodoacetamide (8).…”

Large-scale purification and characterization of the exotoxin of Pseudomonas aeruginosa

“…Analyses of carbohydrate content by gas chromatography were made by using a modification of the method of Bhatti et al (1). Amino acid analyses were performed by a combination of methods currently in use in this laboratory (11). N-terminal amino acids were determined by a dansyl chloride method (30).…”

A novel method for rapid production and purification of exfoliative toxin A of Staphylococcus aureus

“…As a result, the puri¢cation procedure was simpli¢ed from a minimum of six to only three steps because the complication of removing the culture medium proteins and peptides from the toxin preparation was no longer an issue. In addition, reducing the total volume of bacterial cell culture supernatant on the surface of the dialysis sac to only 2^3 ml per £ask (compared to previously reported volumes of up to 50 l [11,14,16,17]) produced a very concentrated crude toxin preparation, which was easier to handle. Two main puri¢cation procedures for the toxins have been described previously^one based on ion exchange chromatography [7,11,12] and the other on isoelectric focusing [8,15^17].…”

“…The described method of toxin production and puri¢cation using a dialysis sac is quicker than previous procedures, taking only a few days instead of weeks [7^17]. Furthermore, the production of 12 mg of crude toxin per ml of bacterial cell culture bathing the surface of the dialysis sac in each conical £ask is much higher than previous reports of 0.01^1.0 mg ml 31 [11,13,14,16,17], as is almost 10 mg puri¢ed toxin per ml of cell culture [11,13,14,16,17] compared to 5^10 mg per 3 l supernatant (1^3 Wg ml 31 ) [17], 100^210 mg per 50 l supernatant (2^4 Wg ml 31 ) [14], and 3^5 mg l 31 (3^5 Wg ml 31 ) [16].…”

“…Di¡erent methods of production and puri¢cation for the exfoliative toxins have been described previously [6^18]. Most procedures require the growth of bacteria in large volumes of culture medium, the puri¢cation time lasts 10 days or more and only small quantities of the ¢nal puri¢ed toxin are produced, usually less than 0.01 mg pure toxin per ml of cell culture supernatant [11,13,14,16,17]. A novel method of high-yield toxin production and puri¢cation using a dialysis sac to separate the culture medium from the staphylococci is described.…”