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.
NIH ExPorter
NIH ExPorter