EXPRESSION OF SUGAR BEET HEMOGLOBIN IN PICHIA PASTORIS

Sammanfattning: People who don’t eat meat can suffer from iron deficiency due to the lower efficiency of absorption of non-heme iron compared to heme iron. The aim of this master thesis was to explore the possibilities of producing a plant-based alternative of heme-iron by evaluating the efficiency of expression of sugar beet hemoglobin in the methylotrophic yeast Pichia pastoris. The gene from sugar beet hemoglobin were inserted into three different Pichia pastoris expression vectors, one for intracellular expression and two for secreted expression using both the native secretion signal from Pichia pastoris and the α-factor signal sequence from Saccharomyces cerevisiae. The three expression vectors were integrated into the Pichia pastoris genome by homologous recombination and the successful transformants were induced with methanol. The expressed proteins were then analyzed using SDS-PAGE. The SDS-PAGE analysis showed no clear evidence of hemoglobin production in Pichia pastoris; however, a more sensitive analysis would need to be performed to determine if hemoglobin was produced or not. Further studies (e.g., ELISA or northern blot) would need to be performed to establish to what degree sugar beet hemoglobin is expressed in the KM71 strain of Pichia pastoris.