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    Engineering an improved expression vector for the Nupl53 RNA binding domain

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    2007_MaertensF_THS_000683.pdf (2.173Mb)
    Author
    Maertens, Francisca
    Advisor
    Jennifer Glowienka; John Addis; Sam Alvey
    Date of Issue
    2007-04-01
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    URI
    https://scholars.carroll.edu/handle/20.500.12647/2916
    Title
    Engineering an improved expression vector for the Nupl53 RNA binding domain
    Type
    thesis
    Abstract
    The nucleoporin Nupl53 has a critical role in both import and export of cellular substances across the nuclear envelope. A unique RNA binding domain has been revealed in Nupl53 (Nupl53-RBD). In order to further understand the interaction between the RNA binding domain (RBD) of Nupl53 and RNA, a crystal structure of the domain would be invaluable. This can be accomplished only if a significant quantity of purified RBD is isolated. Several RBD constructs have been produced to aid in purification of this protein. In most cases, recombinant RBD preparations are fairly heterogeneous and thus unsuitable for structural studies. However, a GFP-RBD construct showed robust expression in E. coli and can be purified without degradation. Although crucial to production and purification, additional domains and tags are not desirable in the final protein sample used for X-ray crystallography. Thus, my goal was to improve the GFP-RBD construct by inserting a protease site that will enable cleavage of the protein once purification is complete, resulting in isolated, pure RBD. Using first QuikChange® and then a PCR based cloning strategy, a TEV protease site was inserted into the GFP- RBD construct. In the future, use of this construct could aide in elucidating how the Nupl53 RNA binding domain recognizes its targets. This could allow us to better understand, and ultimately control, nuclear pore function in the context of normal and disease states.
    Degree Awarded
    Bachelor's
    Semester
    Spring
    Department
    Life & Environmental Sciences
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    • Life and Environmental Sciences Undergraduate Theses

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