Production and application of recombinant biopolymers with exotic chemistries

具有外来化学成分的重组生物聚合物的生产和应用

• 批准号: 9109978
• 负责人: Miriam Amiram
• 金额: $ 5.05万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9109978
• 关键词: Address; Adhesives; Adverse effects; Albumins; Alkynes; Amino Acids; Amino Acyl-tRNA Synthetases; Anti-Bacterial Agents; Antibiotic Resistance; Award; Azides; Back; Bacteria; Bacterial Drug Resistance; Binding; Biocompatible Materials; Biological Process; Biology; Biopolymers; Bypass; Cell Differentiation process; Chemicals; Chemistry; Codon Nucleotides; Communication; Complex; Cues; Data; Development; Differentiation and Growth; Disease; Dopa; Drug Delivery Systems; Drug Kinetics; Elastin; Engineering; Escherichia coli; Eukaryotic Cell; Evolution; Family; Fatty Acids; Formulation; Frequencies; Genome engineering; Goals; Half-Life; Hybrids; Injection of therapeutic agent; Insulin; Libraries; Life; Ligation; Magnetism; Mediating; Mentors; Metals; Methanococcus; Methanosarcina; Methodology; Modeling; Multi-Drug Resistance; Nature; Organism; Peptides; Pharmacologic Substance; Phase; Phase Transition; Phenylalanine; Polymers; Positioning Attribute; Post-Translational Protein Processing; Production; Property; Proteins; Recombinants; Research; Research Training; Resistance; Science; Sense Codon; Silver; Site; System; Technology; Terminator Codon; Therapeutic Agents; Tissues; Training; Transfer RNA; Translating; Translations; Tyrosine; Universities; Variant; Vision; Work; adhesive protein (mussel); antimicrobial; antimicrobial drug; antimicrobial peptide; base; biological systems; biomaterial compatibility; career development; chemical group; clinically relevant; combat; cycloaddition; design; drug resistant bacteria; experience; functional group; glucagon-like peptide; glucagon-like peptide 1; improved; in vivo; nanoparticle; novel; pathogen; peptide drug; polypeptide; public health relevance; pyrrolysine; responsible research conduct; scaffold; skills; synthetic biology

 DESCRIPTION (provided by applicant): Increasingly demanding biomedical and biotechnological endeavors require sophisticated materials that can respond to multiple environmental cues, interact with biological systems, template compound assemblies, and direct the growth and differentiation of cells and tissues. My long term research goal is to create customized and functional protein-based biomaterials containing genetically encoded non- standard amino acids (nsAAs) to site-specifically introduce diverse chemical functionalities. Towards this research vision, my research objectives are two-fold. First, I will work to expand and accelerate our ability to produce protein polymers containing multiple nsAAs. To this end I will develop a novel evolution platform and extensive mutagenized libraries for components used to incorporate nsAAs. Second, I will utilize this platform and variants isolated to introduce novel functions and properties to protein-based biomaterials with the goal of developing antimicrobial and drug delivery formulations. This proposal is designed leverage Prof. Isaacs' expertise in synthetic biology, Prof. Soll's expertise in systems for nsAA incorporation, Prof. Saltzman's expertise in biomaterial-based drug delivery, and Dr. Amiram's previous research experience in protein-polymer synthesis, design and application. In addition to scientific training several career development activities will be pursued during this award including attending career development courses offered by Yale University in science and communication skills, developing mentoring skills, and obtaining extensive training in responsible conduct of research. In our preliminary data, we detail a novel evolution platform for aminoacyl-tRNA synthetases (AARS) for nsAA incorporation. We establish that using evolved AARS variants expressed in a genomically recoded E. coli, we are able to bypass former limitations of nsAA incorporation and achieve high yield and high purity production of biopolymers containing multiple (10-30) nsAA instances. In Aim 1, I will extend and expand AARS libraries to enable evolution of translation components for the incorporation adhesive and bulky nsAAs, which can also be expressed and utilized in prokaryotic and eukaryotic organisms. In Aim 2, I will incorporate multiple azide groups to mediate bioorthogonal attachment of multiple fatty acid molecules to improve peptide and protein pharmacokinetics by enhanced albumin binding. In Aim 3, I will generate adhesive DOPA-biomaterials to create silver-containing formulations for antibacterial treatment. Ultimately, I anticipate that my work will facilitate a fundamental change in our abilities to desin and impart new functions and properties to protein- based materials. The results of these studies will yield enabling technologies and strains for multi-site nsAA incorporation, new classes of biopolymers, customized materials with novel functions, and design rules for manipulating and tuning biopolymer properties.

 描述（由申请人提供）：要求越来越高的生物医学和生物技术工作需要复杂的材料，这些材料可以响应多种环境线索，与生物系统相互作用，模板化合物组装，并指导细胞和组织的生长和分化。我的长期研究目标是创建定制的和功能性的基于蛋白质的生物材料，包含基因编码的非标准氨基酸（nsAA），以特定于位点引入不同的化学功能。为了实现这一研究愿景，我的研究目标是双重的。首先，我将努力扩大和加速我们生产含有多种nsAA的蛋白质聚合物的能力。为此，我将开发一种新的进化平台和广泛的诱变库用于将nsAA的组件。其次，我将利用这个平台和分离的变体，为基于蛋白质的生物材料引入新的功能和特性，目标是开发抗菌和药物递送制剂。该提案旨在利用Isaacs教授在合成生物学方面的专业知识，Soll教授在nsAA掺入系统方面的专业知识，Saltzman教授在生物材料药物递送方面的专业知识，以及Amiram博士以前在蛋白质聚合物合成，设计和应用方面的研究经验。除了科学培训外，该奖项还将开展几项职业发展活动，包括参加耶鲁大学提供的科学和沟通技能职业发展课程，发展辅导技能，并获得负责任的研究行为的广泛培训。在我们的初步数据中，我们详细介绍了一种新的氨酰-tRNA合成酶（阿尔斯）的nsAA掺入的进化平台。我们建立了使用进化的阿尔斯变体在基因组重新编码的E.大肠杆菌中，我们能够绕过nsAA掺入的限制，并实现高产率和高纯度的生产含有多个（10-30）nsAA实例的生物聚合物。在目标1中，我将扩展和扩大阿尔斯库，使翻译组件的掺入粘合剂和庞大的nsAAs，这也可以表达和利用在原核和真核生物的演变。在目标2中，我将引入多个叠氮基团来介导多个脂肪酸分子的生物正交连接，以通过增强白蛋白结合来改善肽和蛋白质的药代动力学。在目标3中，我将生成粘合剂DOPA生物材料，以创建用于抗菌治疗的含银制剂。最终，我期望我的工作将促进我们设计和赋予蛋白质材料新功能和特性的能力的根本变化。这些研究的结果将产生使能技术和菌株，用于多位点nsAA掺入，新类别的生物聚合物，具有新功能的定制材料，以及用于操纵和调节生物聚合物特性的设计规则。