Over-expression of the peptide containing the ATP-binding site in Na/K-ATPase α-subunit

Abstract

The deduced primary structure of Na/K-ATPase from the cloned nucleotide sequence revealed that the structure of ATP-hydrolysing active site was highly conserved among P-type ATPases (1,2,4). However, the minute conformation and molecular mechanism of ion transport have not been made clear yet. X-ray crystallography may be an efficient method to reveal the conformation of the protein, but such a hydrophobic membrane-protein has been unable to be purifed without using detergents. We made an assumption that out-of-membrane domain of Na/K-ATPase α-subunit facing to hydrophilic side with catalytic activity could be crystallized like regular soluble proteins (3). We report here that the hydrophilic, catalytic domain (50kDa) of α-subunit has been over-expressed in E. coli and purified without using any detergent, and discuss on the characterization for the peptide.