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    protaTETHER – a method for the incorporation of variable linkers in protein fusions reveals impacts of linker flexibility in a PKAc‐GFP fusion protein

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    Author
    Norris, Jessica L.; Hughes, Robert M.
    Abstract
    Protein fusions are of fundamental importance in the study of cellular biology and the elucidation of cell signaling pathways, and the importance of linkers for the proper function of protein fusions is well documented in the literature. However, there are few convenient methods available to experimentalists for the systematic implementation of linkers in protein fusions. In this work, we describe a universal approach to the creation and insertion of focused linker libraries into protein fusions. This process, deemedprotaTETHER, utilizes reiterative oligomer design, PCR-mediated linker library generation, and restriction enzyme-free cloning methods in a straightforward, three-step cloning process. We utilize a fusion between the catalytic subunit of cAMP-dependent protein kinase A (PKAc) and green fluorescent protein (GFP) for the development of the protaTETHERmethod, implementing small linker libraries that vary by length, sequence, and predicted secondary structural elements. We analyze the impact of linker length and sequence on the expression, activity, and subcellular localization of the PKAc-GFP fusions, and use these results to select a PKAc-GFP fusion construct with robust expression and enzymatic activity. Based upon the results of both biochemical experiments and molecular modeling, we determine that linker flexibility is more important than linker length for optimal kinase activity and expression.
    Description
    Copyright © 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd. Article licensed under a Creative Commons Attribution License, CC BY 4.0, (https://creativecommons.org/licenses/by/4.0/).
    URI
    http://hdl.handle.net/10342/6813
    Subject
     Linkers; protaTETHER; Protein; Protein engineering; Protein fusions; Restriction enzyme-free; Cloning 
    Date
    2018-03-14
    Citation:
    APA:
    Norris, Jessica L., & Hughes, Robert M.. (March 2018). protaTETHER – a method for the incorporation of variable linkers in protein fusions reveals impacts of linker flexibility in a PKAc‐GFP fusion protein. FEBS Open Bio, (8:6), p.1029-1042. Retrieved from http://hdl.handle.net/10342/6813

    Display/Hide MLA, Chicago and APA citation formats.

    MLA:
    Norris, Jessica L., and Hughes, Robert M.. "protaTETHER – a method for the incorporation of variable linkers in protein fusions reveals impacts of linker flexibility in a PKAc‐GFP fusion protein". FEBS Open Bio. 8:6. (1029-1042.), March 2018. August 16, 2022. http://hdl.handle.net/10342/6813.
    Chicago:
    Norris, Jessica L. and Hughes, Robert M., "protaTETHER – a method for the incorporation of variable linkers in protein fusions reveals impacts of linker flexibility in a PKAc‐GFP fusion protein," FEBS Open Bio 8, no. 6 (March 2018), http://hdl.handle.net/10342/6813 (accessed August 16, 2022).
    AMA:
    Norris, Jessica L., Hughes, Robert M.. protaTETHER – a method for the incorporation of variable linkers in protein fusions reveals impacts of linker flexibility in a PKAc‐GFP fusion protein. FEBS Open Bio. March 2018; 8(6) 1029-1042. http://hdl.handle.net/10342/6813. Accessed August 16, 2022.
    Collections
    • 2017-2018 Open Access Publishing Fund
    • Chemistry

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