Expression, purification, and refolding of the recombinant extracellular domain β1-subunit of the dog Na+/K+-ATPase of the epithelial cells
Protein Expression and Purification(2022)
摘要
The β1-subunit of the Na+/K+-ATPase is a cell membrane protein, beyond its classic functions, it is also a cell adhesion molecule. β1-subunits on the lateral membrane of dog kidney epithelial cells trans-interact with β1-subunits from another neighboring cells. The β-β interaction is essential for the formation and stabilization of intercellular junctions. Previous studies on site-directed mutagenesis and in silico revealed that the interaction interface involves residues 198–207 and 221–229. However, it is necessary to report the interaction interface at the structural level experimentally. Here, we describe the successful cloning, overexpression in E. coli, and purification of the extracellular domain of the β1-subunit from inclusion bodies. Experimental characterization by size exclusion chromatography and DLS indicated similar hydrodynamic properties of the protein refolded. Structural analysis by circular dichroism and Raman spectroscopy revealed the secondary structures in the folded protein of type β-sheet, α-helix, random coil, and turn. We also performed β1-β1 interaction assays with the recombinant protein, showing dimers’ formation (6xHisβ1-β1). Given our results, the recombinant extracellular domain of the β1-subunit is highly similar to the native protein, therefore the current work in our laboratory aims to characterize at the atomic level the interaction interface between EDβ1.
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关键词
Recombinant Na+/K+-ATPase β1-subunit,Inclusion bodies,Refolding by SEC,β1-β1 interaction
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