In vitro characterization of metal-binding and glutathione-binding proteins and their potential use to study transition metal homeostasis in plants
Metadata[+] Show full item record
Protein purification is vital for the characterization of the function, structure, and interactions of a protein.(Berg et al. 2002) Before a specific protein can be identified and characterized, the protein is extracted from a complex mixture and purified. Once the protein is pure, we can determine amino acid sequences, characterize the proteins based on its size, charge, shape, and function, investigate the protein's biological functions, and learn about relationships between proteins and diverse organisms. In order to purify a protein, there are a series of processes that isolate one or a few proteins from a complex mixture. The protein may only be a small fraction of the starting material. (Berg et al. 2002) A few different factors go into consideration when choosing a purification method such as the initial state of the protein mixture and the required sample size. Some of the different purification methods include: chromatography, centrifugation, sonication, filtration, and gel electrophoresis. In this thesis, two different proteins are purified and analyzed using different spectroscopic techniques. The first protein purification described in this thesis is bZIP23. bZIP23 is a known transcription factor for zinc homeostasis, however, the putative metal binding domain has not been characterized. A typical zinc-finger domain either follows a 3cysteine-1histidine or a 2cysteine-2histidine cluster pattern but bZIP23 does not follow this pattern, which makes it an unusual candidate for zincbinding. To characterize the putative metal binding domain (MBD) of bZIP23, it is purified from a bacterial vector and compared to a truncated version of bZIP23 without the putative MBD. Once both versions are expressed and purified from a bacterial plasmid using affinity capture, they are then characterized to learn about molecular weight, which metals are bound, if the proteins are monomers or dimers, as well as the overall structure of the gene. Methods described in this thesis include: polymerase chain reaction, transformation, protein purification, bicinchoninic assay, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and Western blot. Once the protein has been purified methods such as fast performance liquid chromatography and inductively coupled plasma mass spectrometry were used to characterize and compare the full-length and truncated versions of bZIP23. The second part of this thesis describes purifying a protein to monitor FRET changes in response to a metabolite, glutathione. Three enzymes known to bind to glutathione were purified and each one was inserted into three separate FRET-cassettes. Florescence was measured before and after the addition of glutathione in hopes that the fluorescence would increase or decrease due to a protein confirmation change once glutathione was added. The purification methods are very similar for both proteins (bZIP23 and FRET sensor). The main difference is that bZIP23 is tagged with a FLAG-tag and the FRET sensor is tagged with a His-tag.