CORE--HEMOGLOBIN ASSEMBLY

核心--血红蛋白组装

基本信息

批准号：
6564612
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
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-01-01 至 2002-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6564612
关键词：
Escherichia coli biomedical facility hemoglobin hemoprotein biosynthesis human tissue metalloporphyrins protein isoforms protein reconstitution site directed mutagenesis synthetic protein

项目摘要

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.

该核心负责制备人血红蛋白的合成突变体的最后阶段。获取变异蛋白的三个步骤：使用适当的寡核苷酸通过位点特异性诱变在质粒中对球蛋白基因的修饰，该基因在大肠杆菌中的表达以及从表达蛋白质中制备血红蛋白。显然，对于任何给定的变体，第一步是完成一次，而随后的步骤根据需要重复产生足够数量的材料。前两个步骤将在阿诺内博士的指导下在爱荷华大学进行。最后一步将是血红蛋白组装核心的责任。冷冻细胞将从爱荷华大学运送到该核心。如图所述，这些细胞将在两种形式之一上包含球蛋白蛋白。这些蛋白质将从细胞和制备的血红蛋白中提取，这些细胞蛋白包含这些球蛋白。将制备合成突变体，其中含有金属甲素以外的金属酚，或含有金属酚的混合物。将制备不对称杂化血红蛋白，该血红蛋白要么包含两个不同的字母二聚体（混合突变杂交血红蛋白），要么通过化学交联的血红蛋白四聚体来削弱和稳定。