Team:Lund/Model/Hemoglobin/Introduction
Introduction
Background
With the worldwide blood shortage, a high demand for blood substitutes has evolved. One solution, which is independent of todayâs reliance on blood donors, is the use of recombinant hemoglobin based oxygen carriers (rHBOCs). These substitutes consist of concentrated solutions of purified, acellular human hemoglobin which has been heterologously expressed in a non-native host. The advantages over human donated blood are numerous and include universal compatibility, longer shelf life, and reduced risk of disease transmission as well as enhanced oxygen delivery. However, despite the many advantages, rHBOCs are still not being used in practice. This is mainly due to cases of hypertension caused by globin denaturation, misfolding, heme orientational disorder and heme loss. In addition, there are numerous downstream processing problems which includes the removal of lipopolysaccharides (when expressed in e.g. E. coli), protein impurities and free porphyrins.
These issues are mainly caused by the direct injection of free hemoglobin into the bloodstream, of which the environment differs significantly from inside the native red blood cells. As such, many of the issues are related to the interaction of hemoglobin with the surrounding environment, either directly or after a subsequent decomposition. With this, several properties must be modified in order to make hemoglobin a viable blood substitute. This includes the reduction of nitric oxide scavenging as well as reducing oxidative reactivity and increasing the stability of the protein. In addition, the oxygen binding affinity must be lowered to a level appropriate for the new environment.
The approaches to solve the related problems have historically been via rational design strategies in which operative parts of the hemoglobins are mutated. Major achievements include, but are not limited to, the introduction of the di-đŒ mutation which merges the alpha subunits together via a glycine linker, preventing dimeric dissociation, and the V1M mutation which facilitates bacterial translation. However, despite the advances there is still no way of achieving sufficient properties without compromising any other property such has nitric oxide scavenging or oxygen binding affinity. To this end, there is no approved formulation of rHBOCs for the use as a blood substitute.
Source: Cornelius L. Varnado, Todd L. Mollan, Ivan Birukou, Bryan J.Z. Smith, Douglas P. Henderson, and John S. Olson. (2013) âDevelopment of Recombinant Hemoglobin-Based Oxygen Carriersâ. Antioxidants & Redox Signaling Vol. 18, No. 17
Approach
We approach the stated problem in a semi data-driven manner by combing machine learning and qualitative protein design. Our goal is to design hemoglobins by training a learning algorithm to discriminate high- from low-affinity mutants based on previous functional data. Following this, a set of candidates are generated in silico and scored by their likelihood of decreasing the oxygen affinity after being filtered through the algorithm. The high-scoring mutants are then manually selected and evaluated qualitatively based on biological insight and protein engineering principles. By this procedure, the huge number of possible mutants are reduced to a manageable set of rationally chosen candidates which may be subjected for further analysis.
