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    Design, Expression, and Characterization of a Bifunctional Protein Chimera for Applications in Molecular Biology

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    Author
    Taylor, William
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    This item will be available on: 2021-05-01
    Abstract
    There are many different methods of protein capture that are used to immobilize proteins on solid supports while still maintaining their overall function. In this study, the binding interaction between streptavidin and biotin, one of the strongest known non-covalent interactions, is used for the immobilization of a protein kinase onto a solid support. This study is focused on the expression and characterization of a protein fusion between streptavidin and the catalytic subunit of cAMP-dependent protein kinase (PKAcs). The protein fusion will be tested for its expression level, solubility, kinase activity and its biotin binding affinity. A series of linkers between PKAcs and streptavidin will enable testing of the relationship between kinase activity, linker length, and linker flexibility. Upon expression of the protein, the PKAcs streptavidin construct was immobilized on biotinylated superparamagnetic nanoparticles. Initial efforts focused on the expression of our protein fusion in E. coli. These were hampered by low expression levels of our target protein. A new project was undertaken in which the protein fusion between maltose binding protein (MBP), PKAcs, and a streptavidin mutant (traptavidin) was tested. MBP was included to enhance the solubility, whereas traptavidin was included for its enhanced properties versus streptavidin. This construct was successfully created, and current efforts are aimed at testing the application of the construct with nanobeads. The goal of this study is to create reusable nanobeads with specific kinase activity for life science applications.
    URI
    http://hdl.handle.net/10342/7349
    Subject
     protein fusion; immobilization 
    Date
    2019-05-02
    Citation:
    APA:
    Taylor, William. (May 2019). Design, Expression, and Characterization of a Bifunctional Protein Chimera for Applications in Molecular Biology (Honors Thesis, East Carolina University). Retrieved from the Scholarship. (http://hdl.handle.net/10342/7349.)

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    MLA:
    Taylor, William. Design, Expression, and Characterization of a Bifunctional Protein Chimera for Applications in Molecular Biology. Honors Thesis. East Carolina University, May 2019. The Scholarship. http://hdl.handle.net/10342/7349. March 04, 2021.
    Chicago:
    Taylor, William, “Design, Expression, and Characterization of a Bifunctional Protein Chimera for Applications in Molecular Biology” (Honors Thesis., East Carolina University, May 2019).
    AMA:
    Taylor, William. Design, Expression, and Characterization of a Bifunctional Protein Chimera for Applications in Molecular Biology [Honors Thesis]. Greenville, NC: East Carolina University; May 2019.
    Collections
    • Honors College
    Publisher
    East Carolina University

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