Ramotar K, Boyd B, Tyrrell G, Gariepy J, Lingwood C, Brunton J
Samuel Lunenfield Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
Biochem J. 1990 Dec 15;272(3):805-11. doi: 10.1042/bj2720805.
The cistron encoding the B subunit of Escherichia coli Shiga-like toxin I (SLT-I) was cloned under control of the tac promoter in the expression vector pKK223-3 and the SLT-I B subunit was expressed constitutively in a wild-type background and inducibly in a lacIq background. The B subunit was located in the periplasmic space, and less than 10% was found in the culture medium after 24 h incubation. Polymyxin B extracts contained as much as 160 micrograms of B subunit/ml of culture. B subunit was purified to homogeneity by ion-exchange chromatography followed by chromatofocusing. Cross-linking analysis of purified native B subunit showed that it exists as a pentamer. In gels containing 0.1% SDS the native protein dissociated into monomers. B subunit was found to have the same glycolipid-receptor-specificity as SLT-I holotoxin. Competitive binding studies showed that B subunit and holotoxin had the same affinity for the globotriosylceramide receptor. We conclude that this recombinant plasmid is a convenient source of large amounts of purified SLT-I B subunit, which could be used for biophysical and structural studies or as a natural toxoid.
编码大肠杆菌志贺样毒素I（SLT-I）B亚基的顺反子在表达载体pKK223-3中tac启动子的控制下被克隆，并且SLT-I B亚基在野生型背景下组成型表达，在lacIq背景下诱导型表达。B亚基定位于周质空间，培养24小时后在培养基中发现的量不到10%。多粘菌素B提取物每毫升培养物中含有多达160微克的B亚基。通过离子交换色谱随后进行色谱聚焦将B亚基纯化至同质。纯化的天然B亚基的交联分析表明它以五聚体形式存在。在含有0.1% SDS的凝胶中，天然蛋白质解离成单体。发现B亚基与SLT-I全毒素具有相同的糖脂受体特异性。竞争性结合研究表明B亚基和全毒素对球三糖神经酰胺受体具有相同的亲和力。我们得出结论，这种重组质粒是大量纯化的SLT-I B亚基的便利来源，可用于生物物理和结构研究或作为天然类毒素。
