Design of Heme Protein-Based Blood Substitutes

基于血红素蛋白的血液替代品的设计

• 批准号: 7489686
• 负责人: JOHN S. OLSON
• 金额: $ 4.32万
• 依托单位: RICE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-08-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7489686
• 关键词: Adverse effects; Affinity; Amino Acids; Animal Model; Animals; Apoproteins; Bacteria; Basic Science; Binding; Biochemical; Biological Models; Bioreactors; Blood; Blood Banks; Blood Pressure; Blood Substitutes; Blood capillaries; Chromosomes; Chronic; Collaborations; Complement component C1s; Custom; Dependence; Developed Countries; Developing Countries; Development; Disasters; Dissociation; Distal; Drug Formulations; Emergency treatment; Engineering; Erythroid; Escherichia coli; Figs - dietary; Generations; Genes; Globin; Goals; Health Priorities; Heme; Hemeproteins; Hemoglobin; Hemorrhage; Human; In Vitro; Investments; Life; Manufactured Materials; Measures; Military Personnel; Modeling; Modification; Molecular Chaperones; Mutagenesis; Mutation; Myoglobin; Operon; Oxidative Stress; Oxygen; Parents; Pennsylvania; Pharmacologic Substance; Plesiomonas shigelloides; Problem Solving; Production; Property; Protein Denaturation; Protein Engineering; Proteins; Protocols documentation; Public Health; Range; Rate; Rattus; Reaction; Recombinants; Relative (related person); Research Personnel; Resistance; Rice; Series; Shigella dysenteriae; Source; System; Technology; Texas; Therapeutic; Transfusion; Universities; Whole Blood; Wisconsin; apohemoglobin; base; capillary; cellular engineering; commercialization; comparative; cost; crosslink; design; extracellular; improved; in vivo; mutant; oxidation; pathogen; pre-clinical; prevent; programs; prototype; research clinical testing; uptake; vector

Our long-range goal is to develop recombinant human hemoglobin (rHb) as the starting material for manufacturing all globin-based O2 delivery Pharmaceuticals. We have established the key requirements for a rHb-based blood substitute: (a) moderate O2 affinity and large O2 dissociation rate constants for efficient transport in capillaries; (b) significantly reduced rates of NO scavenging to prevent hypertensive side effects; (c) resistance to auto- and chemically-induced oxidation to inhibit oxidative stress; and (d) low rates of heme dissociation to increase shelf-life. We have used the mechanisms governing these properties to design second-generation extracellular rHb-based blood substitutes with more efficient O2 transport and little or no hypertensive side effect in pre-clinical animal studies. However, there is a clear need to engineer third-generation, non-vasoactive rHbs with increased resistance to denaturation, enhanced expression yields, and reduced production costs. We propose to solve these problems by: (1) optimizing O2 binding to and NO scavenging by human rHb without compromising globin stability and resistance to degradative reactions; (2) improving the stability and enhancing production of recombinant hemoglobin in E. coli by (a) rational and comparative mutagenesis to increase the resistance of the apoprotein to unfolding and (b) co- expression of the a and (3 rHb chains with the newly discovered erythroid chaperone, alpha hemoglobin stabilizing protein (AHSP); and (3) facilitating rapid incorporation of exogeneously added heme into newly synthesized globins by co-expression of rHb with the heme utilization genes (hug) from Plesiomonas shigelloides and related pathogens. PUBLIC HEALTH: We are developing recombinant human hemoglobin to replace donated blood as the source material for all protein-based oxygen carriers that are being developed for the treatment of severe blood loss and believe that this effort should be a national public health priority. An effective hemoglobin- based blood substitute will alleviate chronic shortages of whole blood, provide emergency treatment following military and civilian disasters, and allow the use of transfusions in developing countries lacking a safe blood banking system.

我们的长期目标是开发重组人血红蛋白 (rHb) 作为起始材料 制造所有基于球蛋白的 O2 输送药品。我们制定了关键要求 基于 rHb 的血液替代品：(a) 中等 O2 亲和力和大 O2 解离速率常数，可实现高效 毛细血管中的运输； (b) 显着降低 NO 清除率，预防高血压副作用 影响； (c) 抵抗自身和化学诱导的氧化以抑制氧化应激； (d) 低利率 血红素解离以延长保质期。我们已经使用控制这些属性的机制来 设计第二代基于 rHb 的细胞外血液替代品，具有更有效的 O2 运输和 临床前动物研究中很少或没有高血压副作用。然而，显然需要设计 第三代非血管活性 rHb，具有增强的抗变性能力和增强的表达能力 产量，并降低生产成本。我们建议通过以下方式解决这些问题：（1）优化O2结合 人类 rHb 清除 NO 和 NO，而不损害珠蛋白稳定性和抗降解性 反应； (2)通过(a)提高重组血红蛋白在大肠杆菌中的稳定性并提高其产量 合理和比较诱变以增加脱辅基蛋白对解折叠的抗性和（b）共- 表达 a 和 (3 rHb 链以及新发现的红细胞伴侣，α 血红蛋白 稳定蛋白（AHSP）； (3)促进外源添加的血红素快速掺入新的血红素中。 通过 rHb 与来自邻单胞菌的血红素利用基因 (hug) 共表达合成球蛋白 志贺氏菌及相关病原体。 公共卫生：我们正在开发重组人血红蛋白，以替代捐赠的血液作为 正在开发用于治疗严重疾病的所有蛋白质氧载体的源材料 失血，并认为这项努力应该成为国家公共卫生的优先事项。有效的血红蛋白—— 基于血液替代品将缓解全血的长期短缺，提供紧急治疗 军事和民事灾难发生后，并允许在缺乏输血的发展中国家使用输血 安全的血库系统。