Genetic Analysis of Inclusion Body Formation in Phage Infected Bacteria

克隆基因的多肽链在细胞内以非天然聚集体（包涵体）的形式聚集已成为生物技术中的一个难题。已经确定多肽链折叠和聚集的细胞内中间体的少数系统之一是噬菌体P22的耐热三聚体尾尖。突变干扰折叠途径中的中间产物，而不是天然蛋白，在多肽链中心区域的30多个位点上绘制。这些突变体的使用揭示了包涵体是由细胞内折叠途径的早期单链中间体形成的。第二位点抑制子被分离出来，减轻了tsf折叠缺陷。基因内两个附近位置的抑制子似乎通过抑制聚集途径起作用。一旦正确折叠，它们不会影响蛋白质的生物活性。这表明链中的一些局部序列的作用是阻断非通路反应，而不是稳定天然蛋白。这些氨基酸序列信息对克隆蛋白的恢复具有实际意义，对破译氨基酸序列编码蛋白质构象的规律具有理论意义。本研究主要通过制备纯化的tsf、sutsf、tsf / sutsf和野生型蛋白的尿素变性形式，并比较它们在体外的可折叠性，来阐明第二位点抑制因子的作用机制。这些实验将确定抑制因子是通过链内相互作用起作用，还是需要与伴侣蛋白等细胞成分相互作用。

The intracellular accumulation of polypeptide chains from cloned genes as non native aggregates - inclusion bodies- has emerged as a problem in biotechnology. One of the few systems for which intracellular intermediates in polypeptide chain folding and aggregation have been identified is the thermostable trimeric tailspike of bacteriophage P22. Mutations which interfere with intermediates in the folding pathway, rather than the native protein, map at over 30 sites in the central region of the polypeptide chain. Use of these mutants has revealed that the inclusion bodies form from an early single chain intermediate in the intracellular folding pathway. Second site suppressors have been isolated which alleviate the tsf folding defects. Suppressors at two nearby sites within the gene appear to act by inhibiting the aggregation pathway. They do not affect the biological activity of the protein once correctly folded. This suggests that the role of some local sequences in the chain is to block off-pathway reactions rather than to stabilize the native protein. Such amino acid sequence information could be of practical significance in the recovery of cloned proteins and of theoretical significance in deciphering the rules through which amino acid sequences encode protein conformation. This proposal focuses on elucidating the mechanism of action of the second site suppressors by preparing urea denatured forms of the purified tsf, sutsf, tsf / sutsf and wild type proteins and comparing their refolding in vitro. These experiments will define whether the suppressors act through intrachain interactions, or require interactions with cellular components such as chaperonins.