title: Purification of recombinant Shiga-like toxin type I A1 fragment from Escherichia coli.
abstract: Shiga-like toxin I A1 (Slt-IA1) is a RNA N-glycosidase which depurinates a specific adenosine of 28 S eukaryotic rRNA thus inhibiting protein synthesis and ultimately leading to cell death. We have overexpressed this protein in Escherichia coli using a high copy number plasmid and purified the enzyme to homogeneity using a three-step process. Slt-IA1 is released from the periplasm of cells using polymyxin B sulfate, precipitated with ammonium sulfate, and adsorbed to a Matrex Gel Green A dye-ligand agarose column. The enzyme is eluted from the Green A agarose as a single peak with 0.32 M NaCl. Slt-IA1 was purified approximately 1979-fold and routinely gave yields greater than 100%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band with an apparent molecular weight of 28,000. An isoelectric point of 5.1 was determined using analytical isoelectric focusing gels. In an in vitro protein synthesis inhibition assay, 0.02 pM of purified Slt-IA1 inhibited protein synthesis by 50%.
authors.affiliation: Department of Microbiology, Molecular Biology, and Biochemistry, University of Idaho, Moscow 83843.
authors.forename: T M
authors.initials: TM
authors.lastname: Zollman
authors.forename: P R
authors.initials: PR
authors.lastname: Austin
authors.forename: P E
authors.initials: PE
authors.lastname: Jablonski
authors.forename: C J
authors.initials: CJ
authors.lastname: Hovde
mesh_terms: D000818:Animals; D001427:Bacterial Toxins / Q000302:isolation & purification* / Q000494:pharmacology; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods; D003603:Cytotoxins / Q000302:isolation & purification* / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015151:Immunoblotting; D008970:Molecular Weight; D010957:Plasmids; D011112:Polymyxin B; D011500:Protein Synthesis Inhibitors / Q000302:isolation & purification / Q000494:pharmacology; D011817:Rabbits; D012156:Reticulocytes / Q000187:drug effects / Q000378:metabolism; D022622:Shiga Toxin 1
chemical_list: D001427:Bacterial Toxins; D003603:Cytotoxins; D011500:Protein Synthesis Inhibitors; D022622:Shiga Toxin 1; D011112:Polymyxin B