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Ultrafast purification and reconstitution of His-tagged cysteine-lessEscherichia coliF1FoATP synthase

Authors
Journal
Biochimica et Biophysica Acta (BBA) - Bioenergetics
0005-2728
Publisher
Elsevier
Publication Date
Volume
1706
Identifiers
DOI: 10.1016/j.bbabio.2004.09.012
Keywords
  • Escherichia Colif1Foatp Synthase
  • Protein Purification
  • Ni-Nta Resin
  • Reconstitution
  • Acma
  • Steady-State Kinetics
Disciplines
  • Biology

Abstract

Abstract His-tagged cysteine-less F 1F o ATP synthase from Escherichia coli was purified using Ni-NTA affinity chromatography. During the purification procedure the loss of total ATPase activity did not exceed 50%, and the extent of purification was about 80-fold. The purified enzyme was essentially free of other proteins, was highly active in ATP hydrolysis (75 units/mg at pH 8 and 37 °C), and was sensitive to N, N′-dicyclohexylcarbodiimide (70%). Incorporation of F 1F o into soybean liposomes yielded well-coupled and highly active proteoliposomes. The entire procedure, from the disruption of cells by French press to the preparation of proteoliposomes, took only about 8 h. Some improvements in procedures for the estimation of rates of both ATP hydrolysis and ATP-dependent 9-amino-6-chloro-2-methoxyacridine (ACMA) fluorescence quenching are described.

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