Recombinant expression and purification of sugar beet Phytoglobins in Escherichia Coli BL21 (DE3) including metabolic engineering of the heme biosynthesis

Sammanfattning: The protein hemoglobin (Hb) is vital for us humans to ensure enough oxygen replacement in the body. Hb-resembling proteins also exist in plants, these are called phytoglobins (Pgbs). Pgbs are very interesting since they can play a considerable role in the development of blood substitutes and iron supplements for human use. A few years ago, genes that code for Pgb in sugar beet were found and identified. In this thesis, one of these genes, BvPgb1.2, was analyzed further. This was done by bacterial cultivations of E. coli BL21 (DE3) that recombinantly produced the sugar beet Pgb BvPgb1.2. Several cultivations were performed, one with native E. coli cells, one with E. coli cells with wildtype BvPgb1.2 gene (rWt) and three different mutations of this gene (K34C, N7C and E120C). It could be concluded that the rWt and the mutants showed a 1000-folds upregulation of the BvPgb1.2 gene in comparison to the native cells seen in the qPCR measurements. However, when investigating the hemH gene, the expression did not vary significantly between the cultivations. HemH is a key gene in heme biosynthesis. During a δ-ALA gradient experiment, an optimal addition of 1M δ-ALA was found to be 138 μl to a 150 ml cultivation flask since the hemH expression was the highest for this addition. Based on the results found in the SDS-PAGE, the QFF purification can be deemed successful. This indicates a thorough removal of other proteins and a fairly pure Pgb sample was received. Gene knockout of the yfex gene, coding for an enzyme that degrades heme, with CRISPR-Cas9 was planned but not finalized. This was due to obstacles during the experiments. A troubleshooting procedure showed that the likely error source was the ligation between the crRNA and the pCRISPR plasmid. All together, it has been shown that rWt and the mutant Pgbs can successfully be produced and purified. However, more trials are needed to ensure reliable results.