Production and application of recombinant biopolymers with exotic chemistries

 描述(申请人提供)：日益苛刻的生物医学和生物技术工作需要复杂的材料，能够响应多种环境提示，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造定制的、基于蛋白质的生物材料，其中包含基因编码的非标准氨基酸(NsAas)，以定位地引入各种化学功能。对于这一研究愿景，我的研究目标是双重的。首先，我将努力扩大和加快我们生产含有多种nsA的蛋白质聚合物的能力。为此，我将为用于整合nsAAs的组件开发一个新的进化平台和广泛的诱变库。其次，我将利用这个平台和分离的变体将新的功能和特性引入基于蛋白质的生物材料，目标是开发抗菌和药物输送配方。这项建议的设计充分利用了Isaacs教授在合成生物学方面的专业知识、Soll教授在NSAA公司系统方面的专业知识、Saltzman教授在基于生物材料的药物输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，发展指导技能，以及获得负责任的研究行为方面的广泛培训。在我们的初步数据中，我们详细介绍了一个用于NSAA掺入的氨基酰-tRNA合成酶(AARS)的新进化平台。我们证明，使用在基因组重新编码的大肠杆菌中表达的进化的AARS变体，我们能够绕过以前NSAA掺入的限制，并实现包含多个(10-30)NSAA实例的生物聚合物的高产率和高纯度生产。在目标1中，我将扩展和扩展AARS文库，以使融合粘附剂和笨重的nsAAs的翻译成分能够进化，这些翻译成分也可以在原核和真核生物中表达和利用。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，通过增强白蛋白结合来改善多肽和蛋白质的药代动力学。在目标3中，我将生产粘附性DOPA生物材料，以创建用于抗菌治疗的含银配方。最终，我预计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发生根本性变化。这些研究的结果将产生用于多位点NSAA掺入的使能技术和菌株、新的生物聚合物类别、具有新功能的定制材料以及用于操纵和调节生物聚合物性能的设计规则。

 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.