Optimization of Recombinant Phytoglobin Production

Sammanfattning

Hemoglobins, part of the globin superfamily, are heme-containing proteins that are involved in numerous biological processes, primarily oxygen transport. In plants, they are known as phytoglobins and serve diverse roles beyond oxygen transportation, with potential applications as artificial blood substitutes, iron supplements, and plant-based meat alternatives. A class 1 phytoglobin from sugar beet Beta vulgaris (BvPgb 1.2) has emerged as a promising candidate for such uses, but efficient recombinant expression remains a challenge. In this master’s thesis, both BvPgb 1.2 rWT and alanine substituted mutant C86A were expressed in E. coli BL21(DE3) and purified through QFF and BHP using an ÄKTATM Avant system. The project aimed to optimize the expression and purification of BvPgb 1.2, with the additional objective of evaluating whether expression analysis can serve as a viable method for optimizing protein cultivation protocols. δ-ALA and IPTG levels were varied, and expression levels were evaluated using RT-qPCR and complemented by an ocular assessment of the protein’s red pigmentation. RT-qPCR results showed that IPTG induced upregulation in BvPgb 1.2 at both IPTG concentrations, with a greater fold change observed at the lower IPTG concentration. Expression of the investigated heme synthesis gene (hemH) varied slightly between conditions, indicating the need for further investigation. Both the RT-qPCR and ocular assessment pointed to the same optimal conditions with an upregulation (236-fold) of the BvPgb 1.2 gene and upregulation (1.4-fold) of the hemH gene. Additionally, further research is needed to determine which protocol most effectively optimizes the expression of BvPgb 1.2.