Cloning, expression and production of Vicia faba Leghemoglobin B

Abstract: In this thesis a preliminary evaluation of the potential of a leghemoglobin (Lb) for the development of a blood substitute in humans, resembling hemoglobin (Hb) in red blood cells, was conducted. Leghemoglobin B (VfLbB), a gene from Vicia faba, was cloned into a pET-DEST42 vector, using the Gateway™ recombination technology. The recombinant vector containing VfLbB was sequenced and confirmed. Then it was transformed into an Escherichia coli BL21-DE3 strain. Seven shake flasks experiments, with volumes ranging from 250 to 660 ml, were conducted. One experiment was dedicated to constructing a bacterial growth curve and the rest to express VfLbB. In four out of these six protein expression experiments, various parameters related to the induction of VfLbB expression were optimized. The two last shake flasks experiments were conducted with the optimized conditions. After the shake flask experiments, the production was scaled up to a five liter fermenter, where three fermentations were carried out. The first fermentation was dedicated to construct a bacterial growth curve and the other two to express VfLbB. Cells from the optimized shake flask cultivations and from the fermentations were sonicated in order to extract VfLbB. Attempts were done to purify VfLbB by carrying out ion exchange chromatography. Two different media were used: CaptoS (cation exchange) and QFF (anion exchange). Unfortunately, no positive results were obtained indicating that the purification needs to be further optimized.