Purification of recombinant shiga-like toxin type I B subunit.
Shiga-like toxin type I (SLT-I) is a cytotoxin produced by certain strains of Escherichia coli. SLT-I is a bipartite molecule comprised of A (SLT-IA) and B (SLT-IB) subunits. In holotoxin, five B subunits are arranged in a pentameric ring and bind to globotriaosylceramide receptors on the surface of susceptible eukaryotic cells. The SLT-IB pentamer is noncovalently associated with one A subunit that has N-glycosidase activity and ultimately causes the death of targeted cells. Using a previously described overexpression vector, plasmid SBC32, we developed a two-step procedure for the purification of biologically active recombinant SLT-IB. Periplasmic proteins were extracted from E. coli JM105(pSBC32), fractionated by ammonium sulfate precipitation, and separated by isoelectric focusing in a pH 3-10 gradient. SLT-IB was present in fractions with pH values between 5.0 and 6.0, consistent with its reported pI of 5.8. SLT-IB was purified to homogeneity in a second step by native polyacrylamide gel electrophoresis. Purified SLT-IB was characterized for biological and biochemical activity. When analyzed by native polyacrylamide gel electrophoresis, the majority of SLT-IB had an apparent molecular weight of 38,900, consistent with a pentameric subunit association. Chemical cross-linking of SLT-IB with disuccinimidyl suberate resulted in species with molecular weights corresponding to dimeric, trimeric, tetrameric, and pentameric forms of B subunit. SLT-IB was not cytotoxic to Vero cells at concentrations as high as 10 micrograms/ml and protected Vero cells from native SLT-I. Purified SLT-IB maintained its ability to associate with SLT-IA to form holotoxin that exhibited toxicity similar to native toxin.
Austin
P R
PR
Department of Microbiology, Molecular Biology, and Biochemistry, University of Idaho, Moscow, Idaho 83843, USA.
Hovde
C J
CJ
D000645:Ammonium Sulfate; D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D005591:Chemical Fractionation; D002522:Chlorocebus aethiops; D003432:Cross-Linking Reagents; D003603:Cytotoxins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D007525:Isoelectric Focusing; D008970:Molecular Weight; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D022622:Shiga Toxin 1; D014709:Vero Cells
D001427:Bacterial Toxins; D003432:Cross-Linking Reagents; D003603:Cytotoxins; D011994:Recombinant Proteins; D022622:Shiga Toxin 1; D000645:Ammonium Sulfate