Austin P R, Hovde C J
Department of Microbiology, Molecular Biology, and Biochemistry, University of Idaho, Moscow, Idaho 83843, USA.
Protein Expr Purif. 1995 Dec;6(6):771-9. doi: 10.1006/prep.1995.0008.
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.
I型志贺样毒素（SLT-I）是由某些大肠杆菌菌株产生的一种细胞毒素。SLT-I是一种由A（SLT-IA）和B（SLT-IB）亚基组成的双分子。在全毒素中，五个B亚基排列成五聚体环，并与易感真核细胞表面的球三糖神经酰胺受体结合。SLT-IB五聚体与一个具有N-糖苷酶活性的A亚基非共价结合，最终导致靶细胞死亡。我们使用先前描述的过表达载体质粒SBC32，开发了一种两步法来纯化具有生物活性的重组SLT-IB。从大肠杆菌JM105（pSBC32）中提取周质蛋白，通过硫酸铵沉淀进行分级分离，并在pH 3-10梯度下通过等电聚焦进行分离。SLT-IB存在于pH值在5.0至6.0之间的组分中，与其报道的5.8的pI一致。第二步通过非变性聚丙烯酰胺凝胶电泳将SLT-IB纯化至同质。对纯化的SLT-IB进行了生物学和生化活性表征。通过非变性聚丙烯酰胺凝胶电泳分析时，大多数SLT-IB的表观分子量为38,900道尔顿，与五聚体亚基缔合一致。SLT-IB与辛二酸二琥珀酰亚胺酯的化学交联产生了分子量对应于B亚基二聚体、三聚体、四聚体和五聚体形式的物种。SLT-IB在浓度高达10微克/毫升时对Vero细胞无细胞毒性，并保护Vero细胞免受天然SLT-I的侵害。纯化的SLT-IB保持了其与SLT-IA缔合形成全毒素的能力，该全毒素表现出与天然毒素相似的毒性。
