Rapid purification of staphylococcal enterotoxin B by high-pressure liquid chromatography.
The Staphylococcus aureus enterotoxins represent a group of proteins that cause emesis and diarrhea in humans and other primates. We have developed a rapid two-step high-pressure liquid chromatography (HPLC) procedure for purification of staphylococcal enterotoxin B (SEB). Sterile filtrates (2.5 liters) of strain 10-275 were adsorbed directly onto a reversed-phase column (50 mm by 30 cm Delta Pak; 300 A [30 nm], 15 microns, C18). SEB was obtained by using a unique sequential gradient system. First, an aqueous ammonium acetate to acetonitrile gradient followed by an aqueous trifluoroacetic acid (TFA) wash was used to remove contaminants. A subsequent TFA to acetonitrile-TFA gradient eluted the bound SEB. Further purification was obtained by rechromatography on a cation-exchange column. From 35 to 45% of the SEB in starting filtrates was recovered. Analysis by immunoblotting of samples separated on sodium dodecyl sulfate-polyacrylamide gels indicated that HPLC-purified SEB exhibited immunological and biochemical properties similar to those of the SEB standard. Induction of an emetic response in rhesus monkeys showed that the HPLC-purified toxin also retained biological activity.
Strickler
M P
MP
Waters Chromatography Division, Millipore Corporation, Fairfax, Virginia 22030.
Neill
R J
RJ
Stone
M J
MJ
Hunt
R E
RE
Brinkley
W
W
Gemski
P
P
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004768:Enterotoxins / Q000032:analysis / Q000302:isolation & purification* / Q000633:toxicity; D015151:Immunoblotting; D008253:Macaca mulatta; D013211:Staphylococcus aureus
D004768:Enterotoxins; C031818:enterotoxin B, staphylococcal