Genetically Engineered Plants: Improved stress tolerance, growth and facilitated protein purification

University dissertation from Pure and Applied Biochemistry, Chemical Center, University of Lund, P.O. Box 124, S-221 00 Lund, Sweden

Abstract: Heterologous genes were expressed in plants in which the overall goal was to increase the value of the final crop. Various aspects were explored and evaluated, such as improving the stress tolerance, enhancing the availability of oxygen in growing cells and producing rare proteins with increased degrees of purity. In Nature several protective mechanisms against environmental stress have evolved. Water deficit, which is caused mainly by cold, salinity and drought, is in many organisms overcome through an endogenous production of compatible solutes. Compatible solutes are molecules characterised by being dipolar, of low molecular mass and non toxic at high intracellular concentrations. Through expressing the betA gene encoding for a bacterial enzyme choline dehydrogenase (CDH) in tobacco and potatoes, part of the ubiquitous pool of choline was converted to glycine betaine, a compatible solute. In tobacco this resulted in an enhanced salt tolerance and the potato plants displayed improved freezing tolerance. By introducing the vhb gene encoding for a bacterial hemoglobin (VHb) in tobacco, different phenotypic effects were observed such as faster germination and higher final yield, as well as an enhanced production of compounds dependent on oxygen for their synthesis. These compounds were chlorophyll and the secondary metabolite nicotine. Furthermore, preliminary results from transgenic Datura innoxia have shown an increased production the important alkaloid scopolamine. New tailor-made techniques are presented for the purification of transgenic protein, which offer considerable advantages over the conventional methods of purification which are time-consuming and expensive. Affinity tails were genetically fused to the genes encoding galactose dehydrogenase and lactate dehydrogenase. Straight tails were composed of 5 to 6 histidine residues and the affinity tail with an alpha-helix-like structure, His-X3-His-X3-His was constructed, where X represents any amino acid. The fusion proteins were expressed and purified from both bacteria and plants. The purifications were based on immobilised metal affinity chromatography (IMAC) and metal affinity precipitation, where the affinity of the histidine residues for the heavy metal ions was exploited.

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