Ultrafast purification and reconstitution of His-tagged cysteine-less Escherichia coli F1Fo ATP synthase.

Journal article

His-tagged cysteine-less F1Fo 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 degrees C), and was sensitive to N,N'-dicyclohexylcarbodiimide (70%). Incorporation of F1Fo 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.

Authors: Ishmukhametov, R; Galkin, M; Vik, S

• Journal: Biochimica et biophysica acta
• Volume: 1706
• Issue: 1-2
• Pages: 110-116
• Publication date: 2005-01-01
• DOI: 10.1016/j.bbabio.2004.09.012
• ISSN: 0006-3002
• Language: English
• Keywords: Fluorescence; Proteolipids; Electrophoresis, Polyacrylamide Gel; Hydrolysis; Escherichia coli; Histidine; Aminoacridines; Plasmids; Proton-Translocating ATPases; Chromatography, Affinity