Zollman T M, Austin P R, Jablonski P E, Hovde C J
Department of Microbiology, Molecular Biology, and Biochemistry, University of Idaho, Moscow 83843.
Protein Expr Purif. 1994 Jun;5(3):291-5. doi: 10.1006/prep.1994.1044.
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%.
志贺样毒素I A1（Slt-IA1）是一种RNA N-糖苷酶，它能使真核生物28S rRNA的一个特定腺苷脱嘌呤，从而抑制蛋白质合成并最终导致细胞死亡。我们使用高拷贝数质粒在大肠杆菌中过表达了这种蛋白质，并通过三步法将该酶纯化至同质。使用硫酸多粘菌素B从细胞周质中释放Slt-IA1，用硫酸铵沉淀，然后吸附到Matrex Gel Green A染料配体琼脂糖柱上。该酶用0.32M NaCl从Green A琼脂糖上以单峰形式洗脱。Slt-IA1纯化了约1979倍，常规产量大于100%。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳显示一条表观分子量为28,000的单带。使用分析型等电聚焦凝胶测定其等电点为5.1。在体外蛋白质合成抑制试验中，0.02 pM纯化的Slt-IA1可抑制50%的蛋白质合成。
