Engineering H-NOX Domains for Therapeutic Oxygen Delivery

工程化 H-NOX 域用于治疗性氧气输送

• 批准号: 7489330
• 负责人: EMILY E WEINERT
• 金额: $ 4.96万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-10 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7489330
• 关键词: Active Sites; Adverse effects; Aerobic; Affinity; Amino Acids; Bacteria; Binding; Biological Assay; Biomedical Engineering; Blood; Blood Banks; Blood Substitutes; Characteristics; Clinical; Condition; Country; DNA Shuffling; Data; Developed Countries; Developing Countries; Development; Distal; Drug Formulations; Dyes; Engineering; Event; Exhibits; Family; Fluorocarbons; Genes; Goals; Growth; Heme; Hemoglobin; Hydrogen Bonding; Hypoxia; Incubated; Induced Mutation; Infection; Investigation; Kinetics; Lactate Dehydrogenase; Lactate Dehydrogenases; Lasers; Libraries; Ligand Binding; Ligands; Link; Measures; Metabolic; Molecular Weight; Mutagenesis; Mutation; Myoglobin; Nitric Oxide; Operative Surgical Procedures; Oxygen; Plasma; Plasma Proteins; Polymerase Chain Reaction; Property; Protein Family; Proteins; Raman Spectrum Analysis; Rate; Research; Risk; Russia; Scaffolding Protein; Solutions; South Africa; Spectrum Analysis; Standards of Weights and Measures; Structure; Techniques; Testing; Therapeutic; Thermoanaerobacter; Tissues; Trauma; Vascular blood supply; Xylose; autooxidation; base; crosslink; design; desire; directed evolution; disease transmission; flash photolysis; formate C-acetyltransferase; improved; member; mutant; novel; scaffold; sensor; success

DESCRIPTION (provided by applicant): The goal of the proposed research is to bioengineer a temporary oxygen carrier for use as an alternative to banked blood. There is currently a need for blood substitutes because of shortages in the blood supply and potential infection spread through donated blood, especially in countries lacking a clean blood supply. Temporary blood substitutes currently under investigation, based on hemoglobins and perfluorocarbons, have yet to meet with significant regulatory success due to side effects. This project will focus on using members of the Heme Nitric oxide/OXygen (H-NOX) protein family as scaffolds for the development of novel temporary oxygen carriers. Temporary oxygen carriers must exhibit long-term stability under standard storage conditions, stability in plasma, and high selectivity for oxygen over nitric oxide, as nitric oxide scavenging leads to hypertensive side effects. H-NOX proteins display ligand selectivity between oxygen and nitric oxide, making them prime candidates for engineering. Amino acid mutations will be generated in the heme distal pocket to increase the ligand selectivity of these proteins, as well as slow the autooxidation rate of the heme. Directed evolution will also be performed on H-NOX proteins to generate libraries of random mutants with altered ligand binding properties. A selection assay and screen will be developed to identify mutants with the desired oxygen binding characteristics. The evolved H-NOXs will then be subjected to the selection assay and screen and those mutants with improved properties will be fully spectroscopically characterized using resonance Raman and UV-visible spectroscopy, as well as stop-flow and laser flash photolysis to measure ligand binding kinetics. The most promising mutants will be tested for plasma compatibility and chemically modified to increase their stability in plasma. The design of novel blood substitutes is very important due to blood shortages and risk of disease transmission from donated blood. The goal of this research is to develop a new blood substitute, based on a known protein scaffold, that has diminished side effects and improved efficacy as compared to current products.

描述（由申请人提供）：拟议的研究的目的是生物工程师临时氧气载体，以替代存储血液。目前需要血液替代品，因为血液供应短缺和潜在的感染通过捐赠的血液传播，尤其是在缺乏清洁血液供应的国家。由于副作用，目前正在研究的临时血液替代物目前正在研究中，尚未取得显着的调节成功。该项目将着重于使用血红素一氧化氮/氧（H-NOX）蛋白家族的成员作为开发新型临时氧载体的支架。临时氧载体必须在标准储存条件下表现出长期的稳定性，血浆中的稳定性以及对一氧化氮的高选择性，因为一氧化氮的清除会导致高血压副作用。 H-NOX蛋白在氧气和一氧化氮之间表现出配体的选择性，使其成为工程学的主要候选者。氨基酸突变将在血红素远端袋中产生，以提高这些蛋白质的配体选择性，并减慢血红素的自氧化速率。定向进化也将在H-NOX蛋白上进行，以生成具有改变配体结合特性的随机突变体的库。将开发选择分析和筛选，以鉴定具有所需氧结合特征的突变体。然后，进化的H-NOX将进行选择测定和筛选，这些突变体具有改进特性的突变体，将使用共振拉曼和可见的紫外线光谱以及停止流和激光闪光照射以测量配体结合动力学。最有希望的突变体将测试血浆兼容性并化学修饰以提高其在血浆中的稳定性。新型血液替代品的设计非常重要，这是由于血液短缺和捐赠血液传播的风险。这项研究的目的是基于已知的蛋白支架开发一种新的血液替代品，与当前产品相比，副作用降低并提高了疗效。