CORE--HEMOGLOBIN ASSEMBLY

该核心负责制备人类血红蛋白的合成突变体的最后阶段。获得变异蛋白有三个步骤：用合适的寡核苷酸对质粒中的珠蛋白基因进行定点突变，在大肠杆菌中表达。大肠杆菌中表达的蛋白质，以及从表达的蛋白质制备血红蛋白。显然，对于任何给定的变体，第一步完成一次，而后续步骤根据需要重复以产生足够量的材料。前两个步骤将在Arnone博士的指导下在爱荷华州大学进行。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州大学运往该核心。如将描述的，这些细胞将含有两种形式之一的球蛋白。这些蛋白质将从细胞中提取，并制备含有这些球蛋白的血红蛋白。将制备含有除血红素以外的金属卟啉或含有金属卟啉的混合物的合成突变体。将制备不对称杂合血红蛋白，其含有两种不同的α二聚体（混合突变杂合血红蛋白），或两者都将通过化学交联血红蛋白四聚体而稳定化。

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.