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: 7627951
• 负责人: JOHN S. OLSON
• 金额: $ 32.56万
• 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/7627951
• 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; 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; 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; public health priorities; research clinical testing; uptake; vector

DESCRIPTION (provided by applicant): 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 alpha and beta rHb chains with the newly discovered erythroid chaperone, alpha hemoglobin stabilizing protein (AHSP); and (3) facilitating rapid incorporation of exogenously 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.

描述（由申请人提供）：我们的长期目标是开发重组人血红蛋白 (rHb) 作为制造所有基于球蛋白的 O2 输送药物的起始材料。我们已经确定了基于 rHb 的血液替代品的关键要求：(a) 中等的 O2 亲和力和大的 O2 解离速率常数，以便在毛细血管中有效运输； (b) 显着降低 NO 清除率，以预防高血压副作用； (c) 抵抗自身和化学诱导的氧化以抑制氧化应激； (d) 血红素解离率低，可延长保质期。我们利用控制这些特性的机制来设计第二代基于细胞外 rHb 的血液替代品，在临床前动物研究中具有更有效的 O2 运输并且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，以增强对变性的抵抗力、提高表达产量并降低生产成本。我们建议通过以下方法解决这些问题：(1) 优化 O2 与人 rHb 的结合以及人 rHb 的 NO 清除，而不损害珠蛋白稳定性和对降解反应的抵抗力； (2)通过(a)合理和比较诱变以增加脱辅基蛋白对解折叠的抗性以及(b)与新的α和βrHb链共表达，改善大肠杆菌中重组血红蛋白的稳定性并增强其产量。发现红系伴侣、α血红蛋白稳定蛋白（AHSP）； (3)通过rHb与志贺邻单胞菌和相关病原体的血红素利用基因(hug)的共表达，促进外源添加的血红素快速掺入新合成的球蛋白中。公共卫生：我们正在开发重组人血红蛋白，以取代捐献的血液，作为所有基于蛋白质的氧载体的来源材料，这些氧载体正在开发用于治疗严重失血，并相信这项工作应该成为国家公共卫生的优先事项。有效的基于血红蛋白的血液替代品将缓解全血的长期短缺，在军事和民用灾难后提供紧急治疗，并允许在缺乏安全血库系统的发展中国家使用输血。