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


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.


  1. 1.

    Møller J.V., Juul B., le Maire M. 1996. Structural organization, ion transport, and energy transduction of P-type ATPases. Biochim. Biophys. Acta 1286, 1–51.

  2. 2.

    Tang X., Halleck M.S., Schlegel R.A., Williamson P. 1996. A subfamily of P-type ATPases with aminophospholipid transporting activity. Science 272, 1595–1497.

  3. 3.

    Geisler M., Koenen W., Richter J., Schumann J. 1998. Expression and characterization of a Synechocystis PCC 6803 P-type ATPase in E. coli plasma membranes. Biochim. Biophys. Acta 1368, 267–275.

  4. 4.

    Jung K., Tjaden B., Altendorf K. (1997) Purification, Reconstitution, and Characterization of kdpD, the Turgor Sensor of Escherichia coli. J. Biol. Chem. 272, 10847–10852.

  5. 5.

    Berkelman T., Garret-Engele P., Hoffman N.E. 1994. The pacL gene of Synechococcus sp. Strain PCC 7942 encodes a Ca2+-Transporting ATPase. J. Bacteriol. 176, 4430–4436.

  6. 6.

    Lichtner R., Wolf H.U. 1980. Phosphorylation of the isolated high-affinity (Ca2+ + Mg2+) ATPase of the human erythrocyte membrane. Biochim Biophys Acta 598, 472–85.

  7. 7.

    Hsieh W., Pierce W.S., Sze H. 1991. Calcium-pumping ATPases in vesicles from carrot cells. Stimulation by calmodulin or phosphatidylserine, and formation of a 120 kilodalton phosphoenzyme. Plant Physiol 97, 1535–1544.

  8. 8.

    Okorokov L.A., Tanner W., Lehle L. 1993. A novel primary Ca2+-transport system from Saccharomyces cerevisiae. FEBS Lett 216, 573–577.

  9. 9.

    Bowman E.M., Siebers A., Altendorf K. 1988. Bafilomycins: A class of inhibitors of membrane ATPases from microorganisms, animal cells, and plant cells. Proc. Natl. Acad. Sci. USA 85, 7972–7976.

  10. 10.

    Lytton J., Westlin M., Burk S.E., Shull G.E., MacLennan D.H. 1992. Functional comparisons between isoforms of the sarcoplasmic or endoplasmic reticulum family of calcium pumps. J. Biol. Chem. 267, 14483–14489.

  11. 11.

    Hochuli E., Döbeli H., Schacher A. 1987. New metal chelate adsorbenss selective for proteins and peptide containg neighbouring histidine residues. J. Chromatography 411, 177–184.

  12. 12.

    Geisler M., Richter J., Schumann J. (1993) Molecular Cloning of a P-type ATPase Gene from the Cyanobacterium Synechocystis sp. PCC 6803. Homology to Eukaryotic Ca2+-ATPases. J. Mol. Biol 243, 1284–128913.

  13. 13.

    Kaneko T., Sato S., Kotani H., Tanaka A., Azamizu E., Nakamura Y., Miyajima N., Hirosawa M., Sugiura M., Sasamoto S., Kimura T., Hosouchi T., Matsuno A., Muraki A., Nakazaki N., Naruo K., Okumura S., Shimpo S., Takeuchi C., Wada T., Watanabe A., Yamada M., Yasuda M., Tabata S. 1996. Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions. DNA Res. 3, 109–13614.

  14. 14.

    Siebers A., Altendorf K. 1988. The K+-translocating Kdp-ATPase from Escherichia coli. Eur. J. Biochem 178, 131–140.

  15. 15.

    Geisler M., Jakobs B., Richter J., Schumann J. 1996. Expression and Characterization of a Synechocystis PCC 6803 P-type ATPase in E. coli Plasma Membranes. Biochim. Biophys. Acta 1309, 189–193.

  16. 16.

    Clarke D.M., Loo T.W., MacLennan D.H. 1990. Functional Consequences of Mutations of Conserved Amino Acids in the β-Strand Domain of the Ca2+-ATPase of Sarcoplasmic Reticulum. J. Biol. Chem. 265, 14088–14092.

  17. 17.

    Inesi G., Zhang Z., Sagara Y., Kirtley M.E. 1994. Intracellular signaling through long-range linked functions in the Ca2+ transport ATPase. Biophys. Chemistry 50, 129–138.

  18. 18.

    Sarkadi B., Enyedi A., Földes-Papp Z., Gárdos G. 1986. Molecular Characterization of the in Situ Red Cell Membrane Calucium Pump by Limited Proteolysis. J. Biol. Chem. 261, 9552–9557.

  19. 19.

    Carafoli E. 1991. Calcium Pump of the Plasma Membrane. Phys. Rev. 71 129–153.

  20. 20.

    Carafoli E. 1995. Biogenesis: Plasma membrane calcium ATPase: 15 years of work on the purified enzyme. FASEB 8, 993–1002.

  21. 21.

    Heim R., Iwata T., Zvaritch E., Adamo H.P., Rutishauer B., Strehler E.E., Guerini D., Carafoli E. 1992. Expression, Purification, and Properties of the Plasma Membrane Ca2+ Pump and of Its Nterminally Truncated 105-kDa Fragment. J. Biol. Chem. 267, 24476–24484.

  22. 22.

    Lanfermeijer F.C., Venema K., Palmgren M.G. 1997. Purification of heterologous expressed plant plasma membrane H+-ATPase by Ni2+ affinity chromatography. Ann. NY Acad. Sci. 834, 139–141.

  23. 23.

    Papp B., Enyedi A., Kovács T., Sarkadi B., Wuytack F., Thastrup O., Gárdos G., Bredoux R., Levy-Toledano S., Enouf J. 1991. Demonstration of Two Forms of Calcium Pumps by Thapsigargin Inhibition and Radioimmunoblotting in Platelet Membrane Vesicles. J. Biol. Chem. 266, 14593–14596.

  24. 24.

    Laemmli U.K. 1970. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227, 680–685.

  25. 25.

    Villalba J.M., Palmgren M.G., Berberián G.E., Ferguson C., Serrano R. 1992. Functional Expression of Plant Plasma Membrane H+-ATPase in Yeast Endoplamic Reticulum. J. Biol. Chem. 267, 12341–12349.

  26. 26.

    Miroux B, Walker J.E. 1996. Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J. Mol. Biol. 260, 289–298.

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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).

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