Design and Characterization of a Single-chain Hemoglobin

单链血红蛋白的设计和表征

• 批准号: 7981330
• 负责人: SPENCER J ANTHONY-CAHILL
• 金额: $ 27.45万
• 依托单位: WESTERN WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7981330
• 关键词: Achievement; Address; American; Binding; Blood; Blood Banks; Blood Substitutes; Characteristics; Circular Dichroism; Collaborations; Collection; Computer software; Critical Care; Development; Engineering; Equilibrium; Equipment; Globin; Goals; Growth; Hemoglobin; Human; Ligand Binding; Link; Methods; Modification; Mutation; NMR Spectroscopy; Nuclear Magnetic Resonance; Oxygen; Peptides; Performance; Plasmids; Protein Engineering; Proteins; Recombinants; Reperfusion Therapy; Reporting; Research; Rice; Site; Structure; Surface; Surveys; Tandem Repeat Sequences; Therapeutic; Tissues; Transfusion; Variant; base; catalase; design; dimer; flash photolysis; gene cloning; improved; mutant; photolysis; public health relevance; vector

DESCRIPTION (provided by applicant): The long-term goal of this research is the design and synthesis of a single-chain recombinant human hemoglobin (sc-rHb) which binds oxygen reversibly and cooperatively. The sc-Hb design is based on insertion of a circularly permuted ?-globin ("cp?") into a surface loop of human ?- globin to form an ?-cp? dimer. Compared to normal hemoglobin, the proposed sc-rHb could be more easily engineered for optimum performance as a red cell substitute. The development of a sc-rHb molecule would be a significant achievement in protein engineering as well as a step forward in the development of oxygen delivery therapeutics that would be of significant value in critical care. The specific aims of the proposed research are: (1) Optimize expression yields for mutant globins using a combination of altered growth conditions and site-directed mutations shown to improve expression yields for recombinant Hb; (2) Optimize the design of the peptide linker in cp? using computational design methods; (3) Clone these new cp? variants into a coexpression vector that includes a covalently linked tandem repeat of human ?-globin ("di? globin"); (4) Characterize the ligand binding and the structures of the mutant hemoglobins, by established spectroscopic methods (photolysis, stopped-flow, 1-D and 2-D 1H-NMR). Specific aims (1) and (3) will be carried out using standard gene cloning and/or synthesis with plasmids that are available from collaborators. Specific aim (2) will be achieved through collaboration with Prof. Brian Kuhlman (U. N. Carolina) who has expertise in the design of surface loops using RosettaDesign software. Our lab will then clone the gene(s) encoding cp? with the optimized linker sequence, and characterize the structure and function of the new mutant(s). Specific aim (4) will be carried out using specialized equipment in the lab of Prof. John Olson (Rice U.) to determine ligand binding characteristics of globin mutants by stopped-flow/flash photolysis, and equilibrium O2 binding. Our lab will characterize the structures of the mutant globins using near-UV circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopies. PUBLIC HEALTH RELEVANCE: In the 2005 Nationwide Blood Collection and Utilization Survey Report, the American Association of Blood Banks reported on the shrinking margin between the number of available units of blood approved for administration and the number of transfusions. The goal of this research is to address the projected shortfall in blood needed for transfusion, by developing a safe and efficacious red cell substitute based on modification of the human hemoglobin protein.

描述（由申请人提供）：本研究的长期目标是设计和合成一种可逆和协同结合氧的单链重组人血红蛋白（sc-rHb）。sc-Hb设计是基于插入一个循环排列的？-珠蛋白（“cp？“）变成一个人的表面循环？-珠蛋白形成一个？cp？二聚体。与正常血红蛋白相比，所提出的sc-rHb可以更容易地被设计为作为红细胞替代品的最佳性能。sc-rHb分子的开发将是蛋白质工程的一项重大成就，也是氧气输送疗法开发的一个进步，这在重症监护中具有重要价值。建议的研究的具体目标是：（1）优化突变体球蛋白的表达产量，使用改变的生长条件和定点突变的组合，以提高重组血红蛋白的表达产量;（2）优化肽接头的设计在CP？（3）克隆这些新的cp？将一种人β-淀粉样蛋白变体插入共表达载体中，该载体包括一个共价连接的人β-淀粉样蛋白的串联重复序列。珠蛋白（“di？globin”）;（4）通过已建立的光谱方法（光解、停流、1-D和2-D 1H-NMR）表征突变血红蛋白的配体结合和结构。具体目标（1）和（3）将使用标准基因克隆和/或用合作者提供的质粒进行合成。具体目标（2）将通过与Brian Kuhlman教授（美国）的合作实现。N.卡罗莱纳），他拥有使用RosettaDesign软件设计曲面环的专业知识。我们的实验室将克隆编码cp的基因？与优化的接头序列，并表征新突变体的结构和功能。具体目标（4）将使用约翰奥尔森教授（莱斯大学）实验室的专用设备进行。通过停流/闪光光解和平衡O2结合来确定珠蛋白突变体的配体结合特性。我们的实验室将使用近紫外圆二色性（CD）和核磁共振（NMR）光谱来表征突变球蛋白的结构。 公共卫生关系：在2005年全国血液收集和利用调查报告中，美国血库协会报告了批准用于管理的可用血液单位数量与输血数量之间的差距缩小。这项研究的目的是通过开发一种基于人类血红蛋白蛋白修饰的安全有效的红细胞替代品，来解决预计的输血所需血液短缺问题。