A Green Alternative for the Development of Blood Substitutes

Abstract

BvHb1.2 is a sugar beet haemoglobin (Hb) and a good candidate to be used as an artificial oxygen carrier (AOC). 19 mutants of BvHb1.2 were produced with random mutagenesis and expressed in BL-21 E. coli. The Hb content in whole cells was determined by using a refraction index modifier by mixing cell suspensions with 35% BSA. The two mutants (4 and 15) with the highest Hb production were purified using a two-step liquid chromatography; the first step was anion exchange and the second hydrophobic interaction. The rate of autoxidation of both mutants and the wildtype at 22 and 37°C was tested for 9 and 24 hours, respectively. Mutant 15 had a slower rate of autoxidation at 37°C and the same rate at 22°C as the wildtype. Mutant 4 had the same rate of autoxidation at 22°C and at 37°C as the wildtype. Experiments planned to be carried out at the University of Essex were cancelled due to the Covid-19 pandemic.

Popular Abstract

Blood transfusion is a life-saving practice that play an important role in maintaining patients’ essential oxygen delivery system. The practice is heavily dependent on voluntary blood donations, which is a sparse resource in high-income countries and even sparser in middle- and low-income countries.
Artificial oxygen carriers (AOCs) is a potential new solution to end blood shortages in hospitals across the world. In the last 40 years, the focus within AOC research has shifted from true blood substitutes to oxygen therapeutics. While oxygen therapeutics cannot completely replace current blood-based treatments used in hospitals today, they could be used to reduce the amount of blood donations needed.
Haemoglobin-based oxygen carriers (HBOCs) is a type of AOC that has been heavily researched. Plant based haemoglobins (Hbs) are particularly interesting, since they tend to be more stable than human and bovine Hbs. This is due to a slight difference in the binding of the central heme-group at the centre of the protein. This makes them more less prone to autoxidation and therefore more stable. However, plant Hbs bind oxygen much harder than human Hbs and would not release oxygen to even severely oxygen deficient tissue.
The sugar beet has three Hbs that could potentially be used for HBOCs, as they bind relatively weakly to oxygen, compared to other plant Hbs. The sugar beet Hb BvHb1.2 was mutated randomly to produce 20 mutant proteins that were then expressed in E. coli. These mutants were screened for high Hb production. Two mutants, mutant 4 and 15, were then purified, and the rate of autoxidation was then tested.
Mutant 15 had slower rate of autoxidation as the wildtype at 37°C and the same rate at 22°C. Mutant 4 had the same rate of autoxidation as the wildtype at both 22 and 37°C. This suggests that mutant 15 may be a better candidate for further investigations. However, further investigations into the oxygen affinity of the mutants need to be made to understand how the mutations affected the protein.

• author: Hugner, Sara
• supervisor: Nélida Leiva Eriksson
• organization: Pure and Applied Biochemistry; Computational Chemistry
• alternative title: Ett Grönt Alternativ för Utvecklingen av Blodersättning
• course: KBKM05 20201
• year: 2020
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences; Technology and Engineering
• keywords: Applied Biochemistry, Artificial Oxygen Carriers, Haemoglobin, Random Mutagenesis, Error-Prone PCR, Autoxidation, Applied Microbiology, Teknisk mikrobiologi
• language: English
• id: 9027667
• date added to LUP: 2020-09-14 15:55:41
• date last changed: 2020-09-14 15:55:41