Construction of a GFP containing recombinant plasmid facilitating immobilization, visualization and quantification of fusion proteins on interactive polymeric surfaces
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Şahin, Erinç (2003) Construction of a GFP containing recombinant plasmid facilitating immobilization, visualization and quantification of fusion proteins on interactive polymeric surfaces. [Thesis]
Official URL: http://risc01.sabanciuniv.edu/record=b1081778 (Table of Contents)
A two-component system was designed and developed to facilitate immobilization of any peptide such as enzyme or signal peptide onto appropriately modified surfaces. The system was based upon a specialty plasmid that codes a sequence for a poly(6)histidine tag, a GFP (green fluorescent protein) gene, and a multiple cloning site, to which the gene of the target peptide may be inserted, and an interactive surface that was tailored with negatively charged functional groups. The target plasmid bearing a poly(6)histidine coding region was engineered starting from pETM-11 plasmid, which is a vector optimized for expression purposes. In order to better preserve the traits of pETM-11, the non-essential gene of a protein called MAD was excised and replaced with a frame adaptor, as well as GFP gene of comparable size to MAD. This "frame adaptor" - a frame-adjusting, small oligonucleotide sequence joining the histidine anchors and the fluorescent GFP linker - prevented the frameshift problem that would be introduced after substituting the MAD gene with the GFP gene. In addition, it was designed to code for a highly flexible sequence of amino acids (Gly-Gly-Thr) to best preserve the solution-phase traits of the GFP gene. The finalized "immobilization adapter" protein construct, termed GFPimm, was expressed and isolated using either a Ni²+ polyhistidine tag affinity column or ion exchange chromatography. Following isolation, the protein is tested for binding performance using surface-modified polystyrene 96-well plates. In contrast to the typical mode used to bind polyhistidine tags, in which a coordination bond between Ni²+ and imidazole anchors the protein to a surface, the strategy proposed herein was to exploit the positive charges of histidine and negative charges on the surface to achieve salt bridging. For this purpose, a polystyrene surface was modified to bear negatively charged surface groups via incubation with a persulfate reagent, ammonium persulfate. It follows that the recombinant vector designed and constructed in this study is not only amenable to cloning and expression but also for realizing easy purifications, visual tagging of target proteins by fusion methodologies, and performing immobilizations in specialty applications.
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