CORE--HEMOGLOBIN ASSEMBLY

核心--血红蛋白组装

• 批准号: 6410447
• 负责人: ROBERT W NOBLE
• 金额: $ 17.7万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-01-01 至 2001-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6410447
• 关键词: Escherichia coli; biomedical facility; hemoglobin; hemoprotein biosynthesis; human tissue; metalloporphyrins; protein isoforms; protein reconstitution; site directed mutagenesis; synthetic protein

This core is responsible for the final stage in the preparation of synthetic mutants of human hemoglobin. There are three steps in obtaining a variant protein: The modification of the globin gene in the plasmid by site specific mutagenesis using the appropriate oligonucleotide the expression of this gene in E. coli, and the preparation of hemoglobin from the expressed protein. Obviously for any given variant the first step is done once while the subsequent steps are repeated as needed to produce adequate amounts of material. The first two steps will be carried out at the University of Iowa under Dr. Arnone's direction. The last step will be the responsibility of the Hemoglobin Assembly Core. Frozen cells will be shipped from the University of Iowa to this Core. As will be described, these cells will contain globin proteins on one of two forms. These proteins will be extracted from the cells and hemoglobin prepared which contain these globins. Synthetic mutants will be prepared which contain metalloporphyrins other than heme, or which contain a mixture of metalloporphyrins. Asymmetric hybrid hemoglobins will be prepared which either contain two different alphabeta dimers (mixed mutant hybrid hemoglobins) or both will be pared and stabilized by chemically cross-linking the hemoglobin tetramers.

这个核心负责合成人类血红蛋白突变体的最后阶段。获得变异蛋白有三个步骤：用适当的寡核苷酸通过定点突变修饰质粒中的珠蛋白基因，在大肠杆菌中表达该基因，以及从表达的蛋白中制备血红蛋白。显然，对于任何给定的变体，第一步只进行一次，而后续步骤根据需要重复进行，以生产足够量的材料。前两个步骤将在爱荷华大学阿诺恩博士的指导下进行。最后一步将由血红蛋白组件核心负责。冷冻细胞将从爱荷华大学运往This Core。正如将描述的那样，这些细胞将含有两种形式的珠蛋白。这些蛋白质将从含有这些珠蛋白的细胞和制备的血红蛋白中提取出来。合成突变体将被制备成含有除血红素以外的金属卟啉，或含有金属卟啉混合物的合成突变体。将制备包含两个不同的α-β二聚体(混合突变体杂化血红蛋白)的不对称杂化血红蛋白，或者通过化学交联化血红蛋白四聚体来削减和稳定这两种杂化血红蛋白。