A method for the purification of Shiga-like toxin 1 subunit B using a commercially available galabiose-agarose resin.
We describe a procedure for the affinity purification of Shiga toxin 1 subunit B (SLTB) using a commercial galabiose-agarose resin. Recombinant SLTB was purified to 99% homogeneity in a single-step protocol, from the periplasmic extracts of Vibrio cholerae 0395 N1/pSBC54. SDS-PAGE of the affinity purified SLTB showed one band of 8 kDa MW. SLTB purified by this procedure retained its chemical and biological activity as demonstrated by re-binding to the galabiose-agarose resin, and receptor-mediated binding and uptake in Vero cells. The galabiose-agarose resin could isolate roughly 1mg of SLTB/mL of gel. The resin was stable over 3 years and 500 cycles/year of usage. Hence, this method is a straightforward approach to the large-scale preparation of SLTB at a reasonable cost.
Tarragó-Trani
Maria Teresa
MT
Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061, USA.
Storrie
Brian
B
D000818:Animals; D002522:Chlorocebus aethiops; D002846:Chromatography, Affinity / Q000379:methods; D002847:Chromatography, Agarose / Q000379:methods*; D004187:Disaccharides / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D015964:Gene Expression Regulation, Bacterial; D019897:Periplasm / Q000737:chemistry; D021122:Protein Subunits / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism; D022622:Shiga Toxin 1 / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism; D014709:Vero Cells / Q000737:chemistry; D014734:Vibrio cholerae / Q000737:chemistry / Q000235:genetics
D004187:Disaccharides; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D022622:Shiga Toxin 1; C032103:4-O-alpha-D-galactopyranosyl-D-galactose