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    The expression and purification of the regulatory domain of PKG1 alpha

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    Date Issued
    2012
    Author(s)
    Ryu, Kimoon
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    Embargoed until:
    Indefinite
    Permanent Link
    https://hdl.handle.net/2144/12609
    Abstract
    Cyclic guanosine dependent protein kinases play key roles in many functions throughout the body. Three different types of these kinases exist in humans: type one alpha, one beta, and two. The different kinases exist in different locations within the body and also have different substrates that they phosphorylate. Obtaining a pure sample of these kinases has always been challenging due to cyclic nucleotides that co-purify and contaminate the final samples. If cAMP free samples of the kinase were able to be purified, then figuring out the conformation of the enzyme with and without cyclic nucleotides as well as its activation and binding kinetics of the enzymes can be determined. This information will help us to investigate the detailed mechanism of activation of the enzyme and to develop drugs to target these kinases better. Purification of the enzyme is started with growing them in an adenyl ate cyclase deficient Escherichia coli cell line, TP2000, to avoid cyclic nucleotide contaminations. With use of immobilized-metal affinity, ion exchange, and size exclusion chromatography, I successfully purified the regulatory domain of type 1 alpha kinase yielding 490 µL of the protein at a concentration of 2.2 mg/ml that is completely free of cAMP for the first time. The resulting samples will help us to not only measure its affinity to various cyclic nucleotide ligands, but also initiate co-crystal screen with its native ligand, cGMP.
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    Thesis (M.A.)--Boston University PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.
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