Genetic Analysis of Inclusion Body Formation in Phage Infected Bacteria

细胞内积累的多肽链从克隆 基因作为非天然聚集体-包涵体-已经出现， 生物技术的一个问题。 这是为数不多的 多肽链折叠的细胞内中间体， 已经确定的聚集是热稳定的三聚体 噬菌体P22的尾穗。 突变干扰 折叠途径中的中间体，而不是天然的 蛋白质，地图在30多个地点在中央区域的 多肽链 这些突变体的使用表明， 包涵体是由早期单链中间体形成的， 细胞内折叠途径。 第二个位点抑制器 被分离，其减轻了TSF折叠缺陷。 基因内两个邻近位点的抑制因子似乎通过 抑制聚集途径。 它们不影响 一旦正确折叠，蛋白质的生物活性。 这 表明链中某些局部序列的作用是 阻断非途径反应，而不是稳定天然的 蛋白 这样的氨基酸序列信息可以是 在回收克隆蛋白和 理论上的意义，在破译的规则， 氨基酸序列编码蛋白质构象。 这项建议 着重阐明第二个位点的作用机制 通过制备尿素变性形式的纯化的 tsf、sutsf、tsf / sutsf和野生型蛋白质及比较 它们在体外的重折叠。 这些实验将确定 抑制因子通过链内相互作用起作用，或者需要 与细胞成分如伴侣蛋白的相互作用。

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.