A system for production and rapid purification of large amounts of the Shiga toxin/Shiga-like toxin I B subunit.
We have constructed a plasmid expression vector (pSBC32) that encodes the B subunit of Shiga toxin/Shiga-like toxin I under control of the inducible trc promoter. The encoded B subunit is transported to the periplasmic space, allowing single-step purification of milligram amounts of this protein from periplasmic extracts by using receptor analog affinity chromatography. The purified B subunit interacts normally with both polyclonal antiserum to Shiga toxin and a monoclonal antibody specific for B subunit. B subunit purified in this system is pentameric (as in native holotoxin) and biologically active in blocking binding of Shiga holotoxin to HeLa cells. This expression system may allow rapid purification of sufficient amounts of Shiga toxin B subunit to attempt crystallization or to study its efficacy as a vaccine, either by itself or coupled to an appropriate polysaccharide antigen.
Infectious Disease Unit, Massachusetts General Hospital, Boston 02114.
S B
SB
Calderwood
D W
DW
Acheson
M B
MB
Goldberg
S A
SA
Boyko
A
A
Donohue-Rolfe
D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics / Q000302:isolation & purification*; D001428:Bacterial Vaccines / Q000235:genetics; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004768:Enterotoxins / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D010957:Plasmids; D012756:Sheep; D022622:Shiga Toxin 1; D022603:Shiga Toxins
D001427:Bacterial Toxins; D001428:Bacterial Vaccines; D004768:Enterotoxins; D022622:Shiga Toxin 1; D022603:Shiga Toxins