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Expression of a prokaryotic P-type ATPase in E. coli plasma membranes and purification by Ni2+-affinity chromatography

Abstract

In order to characterize the P-type ATPase from Synechocystis 6803 [Geisler (1993) et al. J. Mol. Biol. 234, 1284] and to facilitate its purification, we expressed an N-terminal 6xHis-tagged version of the ATPase in an ATPase deficient E. coli strain. The expressed ATPase was immunodetected as a dominant band of about 97 kDa localized to the E. coli plasma membranes representing about 20–25% of the membrane protein. The purification of the Synechocystis 6xHis-ATPase by single-step Ni-affinity chromatography under native and denaturating conditions is described. ATPase activity and the formation of phosphointermediates verify the full function of the enzyme: the ATPase is inhibited by vanadate (IC50= 119 μM) and the formation of phosphorylated enzyme intermediates shown by acidic PAGE depends on calcium, indicating that the Synechocystis P-ATPase functions as a calcium pump.

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Geisler, M. Expression of a prokaryotic P-type ATPase in E. coli plasma membranes and purification by Ni2+-affinity chromatography. Biol Proced Online 1, 70–80 (1998). https://doi.org/10.1251/bpo9

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Keywords

  • ATPase Activity
  • Plasma Membrane Vesicle
  • Biological Procedure
  • Calcium Pump
  • Total Cell Extract