Hemoglobin, Hemoglobin-based oxygen carrier, Blood substitute
Our long-term goal is to develop a monodisperse high molecular weight hemoglobin-based oxygen carrier (HBOC) for clinical care. One short-term aim is to ligate hemoglobin (Hb) molecules to a dendritic scaffold utilizing “click-chemistry”. Towards this goal, we have genetically modified the C-terminus of one of the α subunits of a di-α globin to contain the S. aureus sortase A recognition sequence (LPETG) and we have expressed the modified globin in E. coli. Here, we demonstrate that these Hbs can be site-specifically functionalized through sortase-mediated ligation of peptides containing dibenzocyclooctyne (DBCO). We further demonstrated proof-of-concept by conjugating an azide-funtionalized peptide with a fluorescent tag, 6-carboxyfluorescein (6-FAM) to Hb(DBCO). Additionally, work has been done to crystallize Hb with a site-specific mutant N108K, which promotes T-state stability of Hb (low oxygen affinity). Cell-free Hb is known to have high oxygen affinity and by determining the structural basis for improving T-state stability in our novel Hbs we hope to design our HBOC with ideal characteristics for reversible oxygen binding. This work establishes that we can functionalize hemoglobin with “click-chemistry” groups such as cyclooctyne, and conjugate that group to an azide with the ultimate goal of decorating azide-functionalized dendrimers with hemoglobin molecules to transport and exchange oxygen in the body.
Huey, Leah, "Development of a Monodisperse Oligomeric Hemoglobin-Based Oxygen Carrier for Acute Blood Replacement Therapy" (2018). WWU Honors Program Senior Projects. 83.
student projects; term papers
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