Recombinant Expression of Novel Wild Type Oat Hemoglobins in Escherichia Coli

Sammanfattning: Hemoglobin is a protein transporting oxygen through our bloodstreams and is as such essential for aerobic life. Hemoglobins also exist in plants, with a wide variety of functions other than oxygen transport. The study of plant hemoglobins could potentially be interesting for the development of synthetic blood substitutes and have also shown promise as an alternative dietary source of the more bioavailable heme-iron, other than conventional meat. Oat has recently been sequenced, which has allowed insight into its inner proteomic workings. In this master thesis, three novel hemoglobins were identified in the oat genome, transformed into Escherichia coli and recombinantly expressed. The project aimed to maximize the recombinant production of these hemoglobins. A high cell density and the addition of a heme-precursor (δ-ALA) were identified as key parameters for effective expression. Analysis by SDS-PAGE of the proteins produced by E. coli failed to unambiguously identify the sought after hemoglobins following expression, however, transcriptomic analysis by RT-qPCR confirmed oat-hemoglobin transcription. These results suggest that the folding of the target proteins was sub-optimal, resulting in unsatisfactory production of the proteins of interest. More work is needed to elucidate why these oat hemoglobins failed to fold properly and if they hold any promise for relevant applications using other methodology or a different recombinant host. This project also confirmed an upregulation (16-fold) of the key heme biosynthesis gene (hemH) upon successful expression of a sugar beet hemoglobin in E. coli.