Argüello, José M.
Na,K-ATPase is a member of the P-type family of ATPases. It transports sodium and potassium ions against their electrochemical gradients across eukaryotic plasma membranes and consequently has a major role in cellular homeostasis. The events that occur on a molecular level in the protein to perform the energy coupled ion transport are largely unknown. To study the structure-function relationships, cysteine-scanning mutagenesis was performed in order to allow the ATPase to be probed with radioactive, spin labeled and fluorescent reagents. The amino acids in the fifth transmembrane fragment were systematically mutated to cysteines in a previous work. In this study, His-tags were attached to the mutants for purification purposes. To this end, His-tag encoding sequence was introduced to the 5' end of cDNA of Na,K-ATPase mutants. The presence of the His-tag insertion was confirmed by restriction enzyme digest and then corresponding constructs were transfected into mammalian COS cells. The resulting proteins were functional as assessed by their capacity to support cell growth.
Worcester Polytechnic Institute
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Chemistry and Biochemistry