NOVEL ROLE FOR CYSTEINE IN PROTEIN FOLDING ASSEMBLY

从重组基因中有效生产蛋白质是一个关键 制药和生物技术行业的问题， 生物化学和生物医学研究。然而，高水平表达的 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，错误折叠导致蛋白水解， 降解或聚集。蛋白质表达的复杂性 真核和原核系统都指出需要更多的 完全理解决定细胞内 和细胞外多肽链聚集。 为了研究折叠和错误折叠的氨基酸决定簇， 噬菌体尾刺蛋白将用作模型系统。的 P22尾穗的折叠途径已得到很好的表征，最近 公开的晶体结构揭示了几种新颖的结构基序。 最近的研究结果表明，存在于细胞中的一个或多个半胱氨酸残基可能是一个潜在的原因。 在折叠和组装过程中激活交叉B片区域 的三聚体。本提案的具体目标是， 活性半胱氨酸残基在折叠和三聚体组装中的作用 使用传统的生物化学技术和定点诱变， 以及改变氧化还原环境。 我们的动机是提供一个 理解折叠、错误折叠、组装和 聚集，这将导致抑制或 控制折页中的非生产性工序。

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.