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

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, deemed protaTETHER, 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 protaTETHER method, 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.

Enzyme Fusions in Biocatalysis: Coupling Reactions by Pairing Enzymes

“…It would be interesting to see whether this is also the case for other enzyme systems than the ones studied so far. To that end, a recent study reported a straightforward PCR method to generate linker libraries for any fusion construct …”

“…[10] Based on these three studies it seems that, in addition to linker composition, the linker length can have ap ronounced effect on the properties of the enzymes.I t would be interesting to see whether this is also the case for other enzymes ystemst han the ones studied so far.T ot hat end, ar ecent study reported as traightforward PCR method to generatel inker libraries for any fusion construct. [54]…”

A bimolecular fluorescence complementation flow cytometry screen for membrane protein interactions

“…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.…”

Oxidoreductase fusions: engineering enzymes for coupled reactions and stability

“…Over the past decades, some progress was made in optimizing large-scale reactions, employing strategies such as biphasic systems, [54] whole cell conversions, [55] and enzyme immobilization. [49,[56][57] Reviews focusing on biocatalysis with BVMOs from prior years are referred to for a broader overview.…”

“…To that end, a recent study reported a straightforward PCR method to generate linker libraries for any fusion construct. [55]…”