Genetic Analysis of Inclusion Body Formation in Phage Infected Bacteria

噬菌体感染细菌中包涵体形成的遗传分析

• 批准号: 8906984
• 负责人: Jonathan King
• 金额: --
• 依托单位: Massachusetts Institute of Technology
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-09-01 至 1992-02-29
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=8906984&HistoricalAwards=false
• 关键词: Genetic; Analysis; Inclusion; Body; Formation

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.

克隆基因的多肽链的细胞内积累是非天然聚集体的 - 包容体 - 已成为生物技术中的问题。 已经确定了多肽链折叠和聚集中细胞内中间体的少数系统之一是噬菌体p22的热稳定尾巴。 干扰折叠途径中的中间体的突变，而不是天然蛋白质，在多肽链的中央区域的30多个位点映射。 这些突变体的使用表明，包含物体来自早期的单链中间体中的包含物体。 已经隔离了第二个位点抑制器，以减轻TSF折叠缺陷。 基因内两个附近位点的抑制剂似乎通过抑制聚集途径起作用。 它们不影响蛋白质一旦正确折叠的生物学活性。 这表明，某些局部序列在链中的作用是阻止朝门反应，而不是稳定天然蛋白质。 这种氨基酸序列信息在克隆蛋白的恢复中可能具有实际意义，并且在解释氨基酸序列编码蛋白质构象的规则方面具有理论意义。 该提案的重点是通过准备纯化的TSF，SUTSF，TSF / SUTSF和野生型蛋白质的尿素变性形式来阐明第二个位点抑制器的作用机理，并比较其体外重新折叠。 这些实验将定义抑制器是通过内部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。