Single-step method for purification of Shiga toxin-1 B subunit using receptor-mediated affinity chromatography by globotriaosylceramide-conjugated octyl sepharose CL-4B.
A new single-step purification method for Shiga toxin (Stx) was developed using receptor-mediated affinity chromatography, in which Gb3Cer (globotriaosylceramide) was conjugated to octyl Sepharose CL-4B as a carrier. This method achieves high yield and high purity in a small column on which Gb3Cer has been immobilized at high density. Using this affinity column, the Stx1 B subunit was purified with homogeneity by a one-step procedure from a crude extract of recombinant Stx1 B subunit-producing Escherichia coli. The purified Stx1 B subunit conserved a natural pentamer structure confirmed by gel filtration and sedimentation equilibrium analysis. Furthermore, the purified Stx1 B subunit was able to bind specifically to Gb3Cer expressed on Burkitt's lymphoma cells. This versatile purification method can be used to isolate various types of natural as well as recombinant Stx, facilitating fundamental studies of human diseases caused by this toxin.
Nakajima
H
H
Department of Pathology, National Children's Medical Research Center, Taishido, Tokyo, Japan.
Katagiri
Y U
YU
Kiyokawa
N
N
Taguchi
T
T
Suzuki
T
T
Sekino
T
T
Mimori
K
K
Saito
M
M
Nakao
H
H
Takeda
T
T
Fujimoto
J
J
D015153:Blotting, Western; D002240:Carbohydrate Sequence; D002846:Chromatography, Affinity / Q000379:methods*; D002850:Chromatography, Gel; D002855:Chromatography, Thin Layer; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D006017:Glycolipids / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism*; D012685:Sepharose / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D022622:Shiga Toxin 1 / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D014281:Trihexosylceramides / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D014407:Tumor Cells, Cultured / Q000378:metabolism; D014461:Ultracentrifugation
D006017:Glycolipids; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D022622:Shiga Toxin 1; C068556:Shiga-like toxin receptor; D014281:Trihexosylceramides; C027176:octyl-sepharose CL-4B; C018549:globotriaosylceramide; D012685:Sepharose