NOVEL ROLE FOR CYSTEINE IN PROTEIN FOLDING ASSEMBLY

从重组基因中高效地生产蛋白质是一个关键 制药和生物技术行业的问题，以及 生物化学和生物医学研究。然而，高水平的表达 克隆的基因并不总是导致成比例的增加 生物活性产品。通常，错误折叠会导致蛋白质分解 降级或聚集。细胞内蛋白质表达的复杂性 真核和原核系统都表明需要更多的 完全理解决定细胞内和细胞内 和胞外多肽链聚集。 研究折叠和错误折叠的氨基酸决定因素，P22 将以噬菌体尾钉蛋白作为模型系统。这个 P22尾钉的折叠途径被很好地描述，最近 已发表的晶体结构揭示了几个新的结构主题。 最近的结果表明，一个或多个半胱氨酸残基存在于 交指B片区在折叠和组装过程中被激活 三聚体的。这项提案的具体目标是 活性半胱氨酸残基在折叠和三聚体组装中的作用 利用传统的生化技术和定点突变， 并通过改变氧化还原环境。我们的动机是提供一个 了解折叠、错误折叠、组装和 聚合将导致更好的抑制或 控制折叠过程中的非生产性过程。

Efficient production of proteins from recombinant genes is a critical issue in the pharmaceutical and biotechnology industries, and in biochemical and biomedical research. However, high level expression of cloned genes does not always lead to a proportional increase in biologically active product. Often, misfolding leads to proteolytic degradation or aggregation. The complex nature of protein expression in both eukaryotic and prokaryotic systems points to the need for a more complete understanding of the factors which determine both intracellular and extracellular polypeptide chain aggregation. To study amino acid determinants of folding and misfolding, P22 bacteriophage tailspike protein will be used as a model system. The folding pathway of P22 tailspike is well characterized and the recently published crystal structure reveals several novel structural motifs. Recent results suggest that one or more cysteine residues present in the interdigitated B sheet region are activated during folding and assembly of the trimer. The specific goals of this proposal are to characterize the role of the active cysteine residues in folding and trimer assembly using traditional biochemical techniques and site-directed mutagenesis, and by altering the redox environment. Our motivation is to provide an understanding of the determinants of folding, misfolding, assembly, and aggregation which will result in an improved ability to suppress or control nonproductive processes in folding.