Improved Approaches to Protein-Protein Coupling and the Efficient Formation of Hemoglobin bis-Tetramers

My research focused on developing new methods for coupling proteins, with an emphasis on producing hemoglobin (Hb) bis-tetramers. These are predicted to be safe and effective alternatives to red cells in transfusions. Based on previous work, it was known that stabilized tetrameric Hb causes increases in blood pressure in circulation, presumably as the result of their extravasation, which leads to scavenging of nitric oxide, the signal for relaxation of the muscles that surround blood vessels. Our initial approach involved optimizing solubility-directed click chemistry based on the copper-catalyzed azide-alkyne cycloaddition (CuAAC). To increase coupling specificity, we developed a double cross-linking approach in which reactive sites on the α-subunits are blocked with a fumaryl cross-linker and with azide installed on a linker specifically bridging the β-subunits. While this approach gives substantial success, the inefficiencies of the CuAAC process with our protein of interest motivated us to improve the process. Thus, strain-promoted alkyne-azide cycloaddition (SPAAC) was successfully adapted for coupling proteins. Using SPAAC, we also developed extended coupling to produce ‘Hb clusters’ with albumin. The resultant Hb-albumin cluster is a breakthrough product that can be scaled with minimal manipulation. We took the project in a new direction using biotin-avidin affinity as the basis for protein-protein coupling. The approach adds a biotin derivative to a cysteine thiol on each β-subunit of Hb. Avidin then scavenges the cysteine biotinylated Hbs by the high affinity biotin-avidin interaction to give the fully functional triple protein HbAvHb. We evaluated the physical and circulatory properties of the products developed here. These approaches not only apply to creating Hb assemblies but also provide a general route to coupling like proteins.Ph.D.2017-11-30 00:00:0