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: 7844991
• 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 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7844991
• 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; 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）作为生产所有基于球蛋白的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，以在毛细血管中有效转运;（B）显著降低NO清除速率，以防止高血压副作用;（c）对自身和化学诱导的氧化的抗性，以抑制氧化应激;和（d）低的血红素解离速率，以增加保质期。我们已经使用这些属性的机制来设计第二代细胞外rHb为基础的血液替代品，更有效的O2运输和很少或没有高血压的副作用，在临床前动物研究。然而，显然需要工程化第三代非血管活性rHb，其具有增加的变性抗性、增强的表达产率和降低的生产成本。我们提出了解决这些问题的方法：（1）优化人rHb与O2的结合和NO的清除，同时不影响球蛋白的稳定性和对降解反应的抗性;（2）提高重组血红蛋白在E.通过（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）α和β rHb链与新发现的红细胞伴侣α血红蛋白稳定蛋白（AHSP）的共表达，在大肠杆菌中表达;和（3）通过rHb与血红素利用基因（hug）的共表达，促进外源血红素快速掺入新合成的球蛋白中来自类志贺邻单胞菌和相关病原体。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，作为所有正在开发用于治疗严重失血的蛋白质氧载体的源材料，并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。

项目成果

Mechanism of Human Apohemoglobin Unfolding.

人类脱辅血红蛋白的展开机制。

• DOI: 10.1021/acs.biochem.6b01235
• 发表时间: 2017
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Samuel,PremilaP;Ou,WilliamC;PhillipsJr,GeorgeN;Olson,JohnS
• 通讯作者: Olson,JohnS

Biophysical and kinetic characterization of HemAT, an aerotaxis receptor from Bacillus subtilis.

• DOI: 10.1529/biophysj.104.047936
• 发表时间: 2005-04
• 期刊: Biophysical journal
• 影响因子: 3.4
• 作者: Wei Zhang;J. Olson;G. Phillips

Correction: Energetics underlying hemin extraction from human hemoglobin by Staphylococcus aureus.

更正：金黄色葡萄球菌从人血红蛋白中提取血红素的能量学基础。

• DOI: 10.1074/jbc.aac120.015267
• 发表时间: 2020
• 期刊: The Journal of biological chemistry
• 作者: Sjodt,Megan;Macdonald,Ramsay;Marshall,JoannaD;Clayton,Joseph;Olson,JohnS;Phillips,Martin;Gell,DavidA;Wereszczynski,Jeff;Clubb,RobertT
• 通讯作者: Clubb,RobertT

Familial secondary erythrocytosis due to increased oxygen affinity is caused by destabilization of the T state of hemoglobin Brigham (ýýýýýýýýýý(Pro100Leu)).

由于氧亲和力增加而导致的家族性继发性红细胞增多症是由血红蛋白 Brigham (Pro100Leu) 的 T 状态不稳定引起的。

• DOI: 10.1002/pro.2130
• 发表时间: 2012
• 期刊: Protein science : a publication of the Protein Society
• 作者: Mollan,ToddL;Abraham,Bindu;Strader,MichaelBrad;Jia,Yiping;Lozier,JayN;Olson,JohnS;Alayash,AbduI
• 通讯作者: Alayash,AbduI

Passively released heme from hemoglobin and myoglobin is a potential source of nutrient iron for Bordetella bronchiseptica.

血红蛋白和肌红蛋白被动释放的血红素是支气管败血博德特氏菌的营养铁的潜在来源。

• DOI: 10.1128/iai.00407-07
• 发表时间: 2007
• 期刊: Infection and immunity
• 影响因子: 3.1
• 作者: Mocny,JeffreyC;Olson,JohnS;Connell,TerryD
• 通讯作者: Connell,TerryD