SALIVARY PROTEINS--MOLECULAR STUDY OF STRUCTURE/FUNCTION

唾液蛋白——结构/功能的分子研究

• 批准号: 2130747
• 负责人: LIBUSE ANNA BOBEK
• 金额: $ 12.03万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-03-15 至 1997-03-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2130747
• 关键词: Candida albicans; Escherichia coli; antibacterial agents; antifungal agents; antiinfective agents; antiviral agents; chimeric proteins; chromatography; circular dichroism; cysteine; gene deletion mutation; molecular biology; molecular cloning; molecular site; mutant; nucleic acid sequence; oligonucleotides; papain; polymerase chain reaction; protease inhibitor; protein purification; protein sequence; protein structure; protein structure function; recombinant DNA; saliva; site directed mutagenesis

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.

人类唾液中发现的分子具有许多至关重要的保护功能 为了口腔健康。 唾液的保护作用在以下方面变得明显： 唾液流量明显减少的个体（口干症或干燥症） 嘴）。 我们研究的长期目标是设计和生产 具有增强的个体和/或嵌合人唾液分子 保护功能并使用它们作为原型来开发 人工唾液。 实现这一目标的一个重要前提是 完整理解结构与功能的关系 单个唾液分子。 突变体的可用性提供了一种 实现这一目标的最有力的方法。 使用 提出分子生物学技术和重组DNA技术 在本申请中，用于生产天然的、变体（突变体）和 异源系统（大肠杆菌）中蛋白质的嵌合形式将 让我们能够研究唾液蛋白的结构及其功能 分子水平并允许合理设计唾液 替代品。 最近，我们获得了编码 几种类型的唾液蛋白，使我们能够进行 拟议的研究。 我们要关注的分子是半胱氨酸蛋白酶抑制剂 （半胱氨酸蛋白酶抑制剂和抗菌/病毒剂）和 组氨酸（抗真菌，特别是抗念珠菌和抗菌 剂），因为它们具有关键的保护功能并且属于 在生化基础上最具有特征的唾液分子。 具体来说，我们建议：1）生产大量的生物 大肠杆菌表达系统中的活性半胱氨酸蛋白酶抑制剂和组氨酸，2) 构建体 半胱氨酸蛋白酶抑制剂和组氨酸突变体将用于绘制 结构域和/或单个氨基酸残基 对于结构完整性和/或生物功能至关重要 这些蛋白质和 3) 构建并产生各种半胱氨酸蛋白酶抑制剂/组氨酸 具有类似或超过生物活性的嵌合蛋白 那些亲本蛋白质。