SALIVARY PROTEINS--MOLECULAR STUDY OF STRUCTURE-FUNCTION

在人类唾液中发现的分子具有许多重要的保护功能 为口腔健康干杯。唾液的保护作用在以下方面变得明显 唾液流量明显减少(口干或干燥)的个人 嘴)。我们研究的长期目标是设计和生产 增强的单个和/或嵌合的人唾液分子 保护功能，并将其用作开发 人造唾液。实现这一目标的一个基本前提是 对结构-功能关系的完整理解 个别唾液分子。突变体的可用性提供了一个 实现这一目标的最有力的方法。对.的使用 分子生物学技术和重组DNA技术提出 在用于生产的本应用中，原生的、变异的(突变体)和 异源系统(大肠杆菌)中蛋白质的嵌合形式将 允许我们研究唾液蛋白质的结构和功能 分子水平和唾液的合理设计 替代品。最近，我们获得了编码基因的重组克隆 几种唾液蛋白质将使我们能够进行 建议进行的研究。我们将关注的分子是半胱氨酸氨基转移酶 (半胱氨酸蛋白酶抑制剂和抗细菌/病毒药物)和 组他汀类药物(抗真菌，特别是抗念珠菌和抗细菌 代理商)，因为它们具有关键的保护功能，并且是 在生物化学的基础上，唾液分子具有最好的特性。 具体地说，我们建议：1)产生大量的生物 原核表达系统中活性半胱氨酸氨基转移酶和组蛋白的构建 半胱氨酸氨基转移酶和组氨酸的突变体将被用来定位 结构域和/或单个氨基酸残基是 对于结构完整性和/或生物功能是必不可少的 这些蛋白质和3)构建和产生各种胱抑素/组蛋白 具有类似或超过生物活性的嵌合蛋白 这些是亲本蛋白质的。

Molecules found in human saliva have many protective functions critical to oral health. The protective qualities of saliva become evident in individuals with markedly decreased salivary flow (xerostomia or dry mouth). The long range goal of our research is to design and produce the individual and/or chimeric human salivary molecules with enhanced protective functions and to use these as prototypes for development of artificial salivas. An essential prerequisite for this goal is the complete understanding of the structure-function relationship of individual salivary molecules. The availability of mutants provides one of the most powerful ways to achieve this objective. The use of molecular biological techniques and recombinant DNA technology proposed in this application for the production the native, variant (mutant) and chimeric forms of proteins in the heterologous system (E coli) will allow us to study the salivary protein's structure and their function at the molecular level and permit a rational design of salivary substitutes. Recently, we have obtained recombinant clones encoding several types of salivary proteins which will enable us to carry out the proposed studies. Molecules that we will concentrate on are cystatins (cysteine proteinase inhibitors and anti-bacterial/viral agents) and histatins (anti-fungal, particularly anti-candidal and anti-bacterial agents), since they have key protective functions and are amongst the best characterized salivary molecules on a biochemical basis. Specifically, we propose to: 1)produce large amounts of biologically active cystatin and histatin in E coli expression system, 2) construct mutants of cystatin and histatin which will be used for mapping the structural domains and/or individual amino acid residues that are essential for the structural integrity and/or biological function of these proteins and 3) construct and produce various cystatin/histatin chimeric proteins with biological activities resembling or exceeding those of the parent proteins.