protaTETHER: A method for the incorporation of linkers in biomacromolecules

Wurz, Anna I; O’Bryant, Collin T.; Hughes, Robert M.

doi:10.1016/bs.mie.2020.10.001

Cited by 2 publications

(2 citation statements)

References 39 publications

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“…As a result, we have demonstrated various methods in which assay limitations can be successfully circumvented, including “release and catch” approaches. Having established this benchmark, future studies could focus on further optimizing the activity of Halo-PKA fusions through linker optimization studies, , potentially improving immobilized enzyme performance through enhancing substrate accessibility. This work will be of utility to those who wish to study phosphoproteomics in cell lysates without the potentially complicating presence of kinase.…”

Section: Discussionmentioning

confidence: 99%

Design, Heterologous Expression, and Application of an Immobilized Protein Kinase

et al. 2022

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Protein kinase A (PKA) is a biologically important enzyme for cell regulation, often referred to as the “central kinase”. An immobilized PKA that retains substrate specificity and activity would be a useful tool for laboratory scientists, enabling targeted phosphorylation without interference from downstream kinase contamination or kinase autophosphorylation in sensitive assays. Moreover, it might also provide the benefits of robustness and reusability that are often associated with immobilized enzyme preparations. In this work, we describe the creation of a recombinant PKA fusion protein that incorporates the HaloTag covalent immobilization system. We demonstrate that protein fusion design, including affinity tag placement, is critical for optimal heterologous expression in Escherichia coli. Furthermore, we demonstrate various applications of our immobilized PKA, including the phosphorylation of recombinant PKA substrates, such as vasodilator-stimulated phosphoprotein, and endogenous PKA substrates in a cell lysate. This immobilized PKA also possesses robust activity and reusability over multiple trials. This work holds promise as a generalizable strategy for the production and application of immobilized protein kinases.

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Section: Discussionmentioning

confidence: 99%

Design, Heterologous Expression, and Application of an Immobilized Protein Kinase

et al. 2022

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“…Therefore, it is important to test different combinations of construct design to find the optimal pair, as the way the fluorophore fragment and the protein of interest are combined plays a role in the efficiency of complex formation 25 . In addition, the correct construct design is also influenced by the choice of the flexible linker sequence between the YFP fragment and the protein of interest, as the linker length and the specific sequence have an influence on the expression, activity and localization of expressed proteins and their complexes 36 , 37 . In a further study, we have compared the YFP signal using two different linkers for the constructive BiFC complex YFP N − AQP0 + YFP C − CaM.…”

Section: Discussionmentioning

confidence: 99%

A bimolecular fluorescence complementation flow cytometry screen for membrane protein interactions

Schmitz

Glas

Neutze

et al. 2021

Sci Rep

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Interactions between membrane proteins within a cellular environment are crucial for all living cells. Robust methods to screen and analyse membrane protein complexes are essential to shed light on the molecular mechanism of membrane protein interactions. Most methods for detecting protein:protein interactions (PPIs) have been developed to target the interactions of soluble proteins. Bimolecular fluorescence complementation (BiFC) assays allow the formation of complexes involving PPI partners to be visualized in vivo, irrespective of whether or not these interactions are between soluble or membrane proteins. In this study, we report the development of a screening approach which utilizes BiFC and applies flow cytometry to characterize membrane protein interaction partners in the host Saccharomyces cerevisiae. These data allow constructive complexes to be discriminated with statistical confidence from random interactions and potentially allows an efficient screen for PPIs in vivo within a high-throughput setup.

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protaTETHER: A method for the incorporation of linkers in biomacromolecules

Cited by 2 publications

References 39 publications

Design, Heterologous Expression, and Application of an Immobilized Protein Kinase

Design, Heterologous Expression, and Application of an Immobilized Protein Kinase

A bimolecular fluorescence complementation flow cytometry screen for membrane protein interactions

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