Engineering H-NOX Domains for Therapeutic Oxygen Delivery

描述（由申请人提供）：拟议研究的目标是生物工程临时氧载体，作为储存血液的替代品。由于血液供应短缺和可能通过捐献的血液传播感染，特别是在缺乏清洁血液供应的国家，目前需要血液替代品。目前正在研究的基于血红蛋白和全氟碳化合物的临时血液替代品，由于副作用尚未在监管方面取得重大成功。本项目将重点利用血红素一氧化氮/氧（H-NOX）蛋白家族成员作为支架，开发新型临时氧载体。临时氧载体必须在标准储存条件下表现出长期的稳定性，在血浆中的稳定性，以及氧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蛋白在氧和一氧化氮之间表现出配体选择性，使其成为工程的主要候选者。血红素远端袋区会产生氨基酸突变，增加这些蛋白质的配体选择性，同时减缓血红素的自氧化速率。定向进化也将在H-NOX蛋白上进行，以产生具有改变配体结合特性的随机突变体文库。一个选择试验和筛选将发展，以确定突变体所需的氧结合特性。进化后的H-NOXs将进行选择分析和筛选，那些具有改进特性的突变体将使用共振拉曼光谱和紫外可见光谱进行全面的光谱表征，以及停止流动和激光闪光光解来测量配体结合动力学。最有希望的突变体将进行等离子体相容性测试，并进行化学修饰以增加其在等离子体中的稳定性。由于血液短缺和捐献血液传播疾病的风险，新型血液替代品的设计非常重要。这项研究的目标是开发一种新的血液替代品，基于一种已知的蛋白质支架，与现有产品相比，它的副作用更小，疗效更高。

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.