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

ECU Author/Contributor (non-ECU co-authors, if there are any, appear on document)

Robert M. Hughes (Creator)

Jessica L. Norris (Creator)

Institution

East Carolina University (ECU )

Web Site: http://www.ecu.edu/lib/

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.

Additional Information

Publication

Other

Norris , J. L. , & Hughes , R. M. (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) , 1029-1042. https://doi.org/10.1002/2211-5463.12414.

Language: English

Date: 2018

Keywords

Linkers, protaTETHER, Protein, Protein engineering, Protein fusions, Restriction enzyme-free, Cloning

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