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 制造所有以珠蛋白为基础的氧气输送药物。我们已经确定了以下关键要求 一种基于重组人血红蛋白的血液替代品：(A)中等的O2亲和力和大的O2解离速率常数，以实现高效 毛细血管转运；(B)显著降低NO清除率，以预防高血压侧 影响；(C)抵抗自动和化学诱导的氧化，以抑制氧化应激；和(D)低比率 血红素的离解以延长货架期。我们已经使用管理这些属性的机制来 设计第二代基于rHb的细胞外血液替代品，具有更高效的氧气转运和 在临床前动物研究中，高血压副作用很少或没有。然而，显然有必要设计 第三代非血管活性rHbs对变性的抵抗力增强，表达增强 产量，并降低生产成本。我们建议通过以下方法来解决这些问题：(1)优化O2结合以 在不损害珠蛋白稳定性和对降解物抵抗力的情况下，人重组人血红蛋白不能清除 (2)通过(A)提高重组血红蛋白在大肠杆菌中的稳定性和产量 合理和相对的突变以增加脱辅基蛋白对去折叠的抵抗力和(B)共- 新发现的红系伴侣α-血红蛋白对α和(3)RHB链的表达 稳定蛋白(AHSP)；以及(3)促进外源添加的血红素快速并入新的 邻单胞菌血红素利用基因(Hug)与重组人血红蛋白的共表达合成珠蛋白 志贺氏菌和相关病原体。 公共卫生：我们正在开发重组人血红蛋白来取代捐献的血液作为 正在开发的所有蛋白质基氧载体的原始材料，用于治疗重症 他认为，这一努力应成为国家公共卫生的优先事项。一种有效的血红蛋白- 基础血液替代品将缓解长期全血短缺，提供紧急治疗 在军事和民事灾难之后，并允许在缺乏药物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.