Design of Heme Protein-Based Blood Substitutes

我们的长期目标是开发重组人血红蛋白（rHb）作为 生产所有基于球蛋白的氧气输送药物。我们已经建立了关键要求， 基于rHb的血液替代品：（a）中等的O2亲和力和大的O2解离速率常数， 毛细血管转运;（B）显著降低NO清除率，以防止高血压侧 影响;（c）抗自身和化学诱导的氧化，以抑制氧化应激;和（d）低利率 血红素分解的方法来延长保质期我们已经使用了控制这些属性的机制， 设计第二代基于细胞外rHb的血液替代品，具有更有效的O2运输， 在临床前动物研究中几乎没有高血压副作用。然而，显然需要设计 第三代，非血管活性rHb，抗变性能力增强，表达增强 产量和降低生产成本。我们建议解决这些问题：（1）优化O2结合， 人rHb清除NO而不损害珠蛋白稳定性和抗降解性 （2）提高重组血红蛋白在E.大肠杆菌（a） 合理的和比较性的诱变以增加脱辅基蛋白对解折叠的抗性，和（B）共- α和β rHb链与新发现的红系分子伴侣α血红蛋白的表达 稳定蛋白（AHSP）;和（3）促进外源添加的血红素快速掺入新的 邻单胞菌血红素利用基因（hug）与rHb共表达合成球蛋白 类志贺菌和相关病原体。 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， 正在开发的用于治疗严重糖尿病的所有基于蛋白质的氧载体的源材料 他说，他认为这一努力应该成为国家公共卫生的优先事项。有效的血红蛋白- 的血液替代品将缓解慢性全血短缺，提供紧急治疗， 在军事和民事灾难之后，允许在缺乏足够的医疗资源的发展中国家使用输血。 安全的血库系统

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.