We previously showed that over production of a fusion protein in which the prion domain of Saccharomyces cerevisiae [PSI(+)] is connected to glutathione S-transferase (GST-Sup35NM) causes a marked decrease in the colony forming ability of Escherichia coli strain BL21 after reaching stationary phase. Evidence indicated that the observed toxicity was attributable to intracellular formation of fibrous aggregates of GST-Sup35NM. In this report, we describe the isolation of plasmids that encode mutant forms of GST-Sup35NM which do not confer the toxicity to E. coli strain BL21. Each of the four spontaneous mutant-forms of GST-Sup35NM obtained revealed amino acid substitutions. One substitution was located in the N domain, and the others in the M domain. Congo red binding assay indicated that none of these mutant proteins underwent conformational alteration in vitro. From these results, we conclude that the M domain, in collaboration with the N domain, plays an essential role in aggregation of Sup35NM. In addition, our data demonstrate the usefulness of the E. coli expression system in studying aggregate-forming proteins.

中文翻译：

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酵母朊病毒 [PSI+] 突变对大肠杆菌 BL21 菌株中表现出的淀粉样蛋白毒性的影响。

我们之前表明，过度生产一种融合蛋白，其中酿酒酵母 [PSI(+)] 的朊病毒结构域与谷胱甘肽 S-转移酶 (GST-Sup35NM) 连接，导致大肠杆菌菌株 BL21 的集落形成能力显着降低达到稳定期后。证据表明，观察到的毒性可归因于 GST-Sup35NM 纤维聚集体的细胞内形成。在本报告中，我们描述了编码 GST-Sup35NM 突变形式的质粒的分离，这些质粒不会对大肠杆菌菌株 BL21 产生毒性。获得的 GST-Sup35NM 的四种自发突变形式中的每一种都显示了氨基酸取代。一个替换位于 N 域中，其他替换位于 M 域中。刚果红结合试验表明，这些突变蛋白在体外均未发生构象改变。从这些结果中，我们得出结论，M 域与 N 域合作，在 Sup35NM 的聚合中起着至关重要的作用。此外，我们的数据证明了大肠杆菌表达系统在研究聚集形成蛋白方面的有用性。