Large-scale production and purification of <i>Clostridium perfringens</i> type A enterotoxin

Reynolds, David S.; Tranter, Howard S.; Capel, B. J.; Bartholomew, Barbara; Stringer, M. F.

doi:10.1042/bst0120262a

Cited by 1 publication

(3 citation statements)

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“…However, the m e t h o d is occasionally unsuccessful for purification .of the enterotoxin (12,21 ) and the final preparation is contaminated with minor nontoxin proteins (22). These troubles have been attributed to difficulty in maintaining high toxin producing ability of the bacterial strains, resulting in low toxin contents of the starting materials for purification, and limited resolution on conventional gel permeation through a Sephadex G-200 column (12,21).…”

Section: Discussionmentioning

confidence: 99%

“…The toxin is produced during sporulation (7,16) and it has been purified (1,8,12,(20)(21)(22)(23)(24)27) from sonic extracts of sporulated bacteria and shown to be a simple protein with a molecular weight of ca. 35000 (11,13,14,25).…”

Section: Introductionmentioning

confidence: 99%

“…However, there are some problems in this method. Reynolds et al (21) reported that the method gives a variable yield of toxin. Granum and Whitaker (12) found that the method was not effective when the enterotoxin concentration of the crude extract was less than 8 to 10% of the total protein.…”

Section: Introductionmentioning

confidence: 99%

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Purification by high performance liquid chromatography of Clostridium perfringens type a enterotoxin prepared from high toxin producers selected by a toxin-antitoxin halo

1985

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High enterotoxin-producing substrains of Clostridium perfringens type A were selected reproducibly as colonies having toxin-antitoxin haloes on agar plates of Duncan-Strong medium containing antitoxin serum. Enterotoxin from these substrains was subjected to rapid purification by high performance liquid chromatography (HPLC). For this, the toxin was extracted by sonication from sporulating bacteria grown in Duncan-Strong sporulation medium, fractionated by ammonium sulfate (40% saturation) precipitation and differential solubilization and then purified by HPLC: gel permeation chromatography through a G2000SW column and ion-exchange chromatography on a Mono Q column. Purified toxin preparations had a similar specific activity (4.2 X 10(2) mouse MLD/mg protein) and homogeneity on polyacrylamide gel-electrophoresis to preparations obtained by conventional gel permeation through a Sephadex-G200 column. By further HPLC on a Mono Q column, minor nontoxin proteins were separated from the toxin without loss of the toxicity on a protein basis. The final yield of the purified toxin was about 15% of that in the bacterial extract. The two HPLC procedures each took only one hour.

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Section: Discussionmentioning

confidence: 99%