SALIVARY PROTEINS--MOLECULAR STUDY OF STRUCTURE-FUNCTION

在人类唾液中发现的分子具有许多保护功能， 口腔健康。 唾液的保护作用在 唾液流量明显减少的个体（口腔干燥或干燥 口）。 我们研究的长期目标是设计和生产 所述具有增强的唾液分泌的个体和/或嵌合人唾液分子 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先决条件是 完全理解结构-功能关系 单个唾液分子。 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。 使用 提出了分子生物学技术和重组DNA技术 在用于生产的本申请中，天然的、变体（突变体）和 异源系统（大肠杆菌）中的嵌合形式的蛋白质将 使我们能够研究唾液蛋白的结构和功能， 并允许唾液的合理设计 替代品 最近，我们已经获得了重组克隆， 几种类型的唾液蛋白，这将使我们能够进行 建议的研究。 我们将重点关注的分子是胱抑素 （半胱氨酸蛋白酶抑制剂和抗细菌/病毒剂）和 组胺素（抗真菌，特别是抗念珠菌和抗细菌 因为它们具有关键的保护功能， 最好的唾液分子的生化基础上。 具体来说，我们建议：1）生产大量的生物 在大肠杆菌表达系统中表达活性半胱氨酸蛋白酶抑制剂和组蛋白酶抑制剂，2）构建 半胱氨酸蛋白酶抑制剂和组蛋白酶抑制剂的突变体，其将用于绘制 结构域和/或单独的氨基酸残基， 对结构完整性和/或生物功能至关重要 这些蛋白和3）构建和产生各种胱抑素/组胺素 嵌合蛋白，其生物活性类似于或超过 那些母体蛋白质。

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.