SEQUENCING N-TERMINALLY BLOCKED PROTEINS

蛋白质和肽的自动Edman降解是至关重要的 在提供氨基酸序列信息用于鉴定和 对含有相应结构基因的DNA克隆进行测序。 的 包括蛋白质/肽样品制备的复杂程序， 测序化学本身和PTH氨基酸鉴定已经成为 一项不断改进以提高灵敏度的高科技 和适用范围，因此丰度较低的蛋白质和 可以分析日益多样化的特性。 目前的 该技术最重要的限制是， 蛋白质通过N-α-酰化被阻止Edman降解， N-末端残基。 这一限制的意义来自于 从N-末端获得的序列信息的重要性 用于基因克隆和测序研究。 虽然 内部氨基酸序列信息通常可以从 阻止蛋白质，它的收购总是消耗相当大的 蛋白质的数量比N-末端序列分析，以及缺乏一个 N-末端序列随后使得DNA序列数据更加困难 获取和解释。 该提案寻求建立用于解块皮摩尔的通用方法 大量的蛋白质，以便可以进行高灵敏度氨基酸测序， 用于确定N-末端序列。 一个程序，使小 易于被特定解封闭酶附着的蛋白质量 将寻求在工作蛋白质化学中实施 实验室 该程序将基于共价固定的 在与Edman相容固体支持物上的底物蛋白 测序 尽管潜在合适的N α-乙酰化氨基酸 已知水解将解封闭小肽底物， 建议是高风险的，因为他们没有被证明工作更长的时间 多肽。 然而，该项目的成功将导致大量的 氨基酸序列分析灵敏度的提高 大多数蛋白质，并将代表一个重大的改善， 该技术在分子生物学中的实用性。

Automated Edman degradation of proteins and peptides is of crucial importance in providing amino acid sequence information for identifying and sequencing DNA clones that contain the corresponding structural genes. The complex of procedures encompassing protein/peptide sample preparation, the sequencing chemistry itself, and PTH amino acid identification, has become a high technology that is constantly being modified to improve sensitivity and range of applicability, so that proteins of lower abundance and increasingly diverse properties can be analyzed. At the present time, the most important limitation to the technology is that approximately 80% of proteins are blocked tot he Edman degradation by Nalpha- acylation of the N-terminal residue. The significance of this limitation derives from the preeminent importance of sequence information acquired from the N-terminus of the intact protein for gene cloning and sequencing studies. Although internal amino acid sequence information can generally be acquired from blocked proteins, its acquisition always consumes considerably larger amounts of protein than an N-terminal sequence analysis, and the lack of an N-terminal sequence subsequently makes the DNA sequence data more difficult to obtain and interpret. This proposal seeks to establish general methods for deblocking picomole amounts of proteins so that high sensitivity amino acid sequencing can be used to determine the N-terminal sequence. A procedure for rendering small amounts of protein susceptible to attach by specific deblocking enzymes will be sought for implementation in a working protein chemistry laboratory. The procedure will be based on covalent immobilization of the substrate protein on a solid support that is compatible with Edman sequencing. Although potentially suitable Nalpha - acetylation amino acid hydrolyses are known that will deblock small peptide substrates, the proposal is high risk because they have not been shown to work on longer polypeptides. However, success of the project would result in a large improvement in the sensitivity of amino acid sequence analysis of the majority of proteins and would represent a major improvement in the usefulness of the technology in molecular biology.