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: 7144869
• 负责人: JOHN S. OLSON
• 金额: $ 33.53万
• 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/7144869
• 关键词: Escherichia coli; Vibrionaceae; bacterial genetics; biomaterial development /preparation; bioreactors; biotechnology; blood /plasma substitute; conformation; free radical scavengers; hemoglobin; hemoprotein; human genetic material tag; laboratory rat; microspectrophotometry; molecular assembly /self assembly; molecular chaperones; nitric oxide; oxidative stress; oxygen tension; oxygen transport; protein engineering; recombinant proteins; vasoconstriction

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）：我们的远程目标是开发重组人血红蛋白（RHB）作为制造所有基于Globin的O2递送药物的起始材料。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亲和力和较大的O2解离速率常数，用于毛细管的有效运输； （b）显着降低了没有清除率以防止高血压副作用； （c）对自身和化学诱导的氧化的耐药性抑制氧化应激； （d）血红素解离率低以增加保质期。我们已经使用了管理这些特性的机制来设计第二代基于RHB的血液替代物，具有更有效的O2运输，并且在临床前动物研究中很少或没有高血压副作用。但是，显然需要设计第三代，非毒性RHB，具有增强性的耐药性，增强的表达产率和降低的生产成本。我们建议通过以下方式解决这些问题：（1）在不损害球蛋白稳定性和对降解反应的抵抗力的情况下，优化人类RHB与人类RHB的结合，没有清除； （2）通过（a）合理和比较诱变提高大肠杆菌中重组血红蛋白的稳定性和增强的产生发现了红细胞血红蛋白稳定蛋白（AHSP）的红细胞伴侣； （3）通过将RHB与血红素利用基因（HUG）共表达，从shigelloides及其相关病原体的血红素利用基因（HUM）促进新近合成的球蛋白中的快速融合到新合成的环球奥中。公共卫生：我们正在开发重组人血红蛋白，以取代捐赠的血液作为所有基于蛋白质的氧气载体的原始材料，这些氧气载体正在为治疗严重失血而开发，并认为这项工作应该是国家公共卫生的重点。有效的血红蛋白血液替代品将减轻全血的长期短缺，在军事和平民灾难后提供紧急治疗，并允许在缺乏安全的血液银行体系的发展中国家使用输血。