Metal Affinity Peptides in Biotechnological Applications As tools in protein purification, protein stabilisation, metal quantification and metal tolerance of bacteria and plants

Detta är en avhandling från Center for Chemistry and Chemical Engineering

Sammanfattning: Metal affinity tags have been fused to proteins and demonstrated to function as tools in protein purification, protein stabilisation, quantitative metal analysis and metal tolerance of bacteria and plants. In biotechnological applications proteins frequently have to function in nonnatural environments with harsh conditions which impose requirements for high stability on the proteins. Metal affinity tags fused to the N-terminal of lactate dehydrogenase increased the stability of the enzyme under thermal and urea denaturing conditions in the presence of metal ions. Metal affinity tags have frequently been used in purification of proteins with immobilised metal affinity chromatography. The most commonly used tag contains six consecutive histidines. Other tags comprising less histidines may offer less impact on protein activity, stability and expression level. Moreover, a higher degree of selectivity and thus purity may be obtained. A tag folding into an a-helix with surface exposed histidines (His-X3-His-X3-His), where X is any amino acid, was fused to the enzyme lactate dehydrogenase and expressed in tobacco. The amount of lactate dehydrogenase produced in transgenic tobacco was increased as compared to a His6 tagged enzyme. Plants can be used as bioreactors for the production of technical and industrially interesting enzymes. The applicability of producing enzymes in plants is partly determined by the availability of a good purification method. His tagged lactate dehydrogenase was produced in transgenic tobacco and the enzymes could be purified from a crude extract with metal affinity chromatography and metal affinity precipitation. His tagged glucose isomerase was expressed in transgenic potato tubers and the fructose levels were increased in the tubers. Pollution of heavy metals is an increasing environmental problem. The use of micro-organisms and plants for bioremediation has been proposed as an alternative to conventional methods. By expressing metal binding proteins and peptides in bacteria and plants the metal tolerance or accumulation of the organism can be increased. A cadmium binding hexapeptide, His-Ser-Gln-Lys-Val-Phe, was selected from a phage display peptide library. Expression of this peptide on the surface or intracellularly increased the metal tolerance of Escherichia coli. The cadmium binding peptide fused to the N-terminal of green fluorescent protein also endowed tobacco plants with an increased cadmium tolerance. Metal affinity tags fused to green fluorescent protein was demonstrated to function in quantification of transition metal (nickel, copper and zinc) and cadmium ions. The cadmium binding peptide and a His6 tag were fused to the N-terminal of green fluorescent protein and the amount of metal ions could be determined as the relative decrease in fluorescence intensity of green fluorescent protein.

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