Metal Binding Tags - Characterisation, Use in Bioseparation and Applications of Green Fluorescent Fusion Proteins
Sammanfattning: Most of the recombinant proteins produced nowadays are fused to affinity tags, in order to facilitate their purification through affinity chromato-graphy. Out the different tags available, poly-histidine tags are among the most commonly used and can help purification through immobilised metal ion affinity chromatography (IMAC). In this thesis, we present different metal binding tags, and their affinity towards metals ions was tested using different techniques, such as surface plasmon resonance (SPR), IMAC, immobilised metal affinity gel electrophoresis (IMAGE) or partitioning (IMAP). SPR was used to compare the binding strength of different poly-histidine tagged lactate dehydrogenases. The technique proved to be fast and effective, and presented also the advantage to only consume small amounts of material. A peptide library, fused to the green fluorescent protein (GFP), was screened to search for novel metal binding tags, using IMAC in a 96-well plate format and IMAGE. The two methods had their advantages and drawbacks, but in both cases the fusion to GFP was helpful, as it simplified and speeded up the detection of the recombinant proteins. Affinity tags containing both histidine and hydrophobic residues were found to be very efficient in IMAP, using a PEG/salt aqueous two-phase system. Different amino acid combinations were tested and, for the same number of histidines, the partition coefficient of some of the protein constructs was increased more than twice, reaching K values over 20, when hydrophobic residues were introduced in the tag sequence. Finally, chimeric fusion proteins combining Fc-binding and fluorescent properties, were evaluated as alternatives to secondary conjugated-antibodies. GFP was fused to one or two repeats of the Z-domain of protein A (Staphylococcus aureus) and expressed in E. coli. His-tagged variants were also prepared, in order to be purified using IMAC, offering a cheap purification method. All constructs were tested in a fluorescent immunoassay test and proved to be as efficient as a commercial fluorescent antibody conjugate.
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