Reconstitution of the F1-ATPase activity from purified α, β γ and δ or ϵ subunits with glutathione S-transferase fused at their amino termini

Abstract

Systems for overexpression and purification of active alpha, beta and gamma subunits of Escherichia coli H(+)-ATPase were established. The alpha and beta subunits recovered as soluble form were purified by hydroxyapatite column chromatography. Since the gamma subunit was overexpressed as the insoluble form, this subunit was purified by polyacrylamide gel-electrophoresis containing sodium dodecyl sulfate. By subsequent denaturation of this subunit with guanidine hydrochloride and renaturation, the active gamma subunit for reconstitution of the F1-ATPase activity with the purified alpha and beta subunit was obtained. The delta and epsilon subunits which were fused to the carboxy terminus of glutathione S-transferase (GST) were overproduced and purified by affinity chromatography. These fused proteins (delta-GST and epsilon-GST) were incubated with the purified alpha, beta and gamma subunits and applied to affinity chromatography. The alpha beta gamma delta-GST and alpha beta gamma epsilon-GST complex were eluted specifically by addition of glutathione and exhibited high and low ATPase activity, respectively, with a subunit stoichiometry similar to that in the native F1-ATPase, indicating that active complexes could be reconstituted with the fused proteins. These results suggested that the amino-terminal ends of the delta and epsilon subunits are not involved in formation of the active complex. The fused epsilon-GST bound the gamma subunit strongly, and the alpha subunit weakly. The delta-GST bound the gamma subunit significantly, and the alpha and beta subunits very weakly.

Calcineurin Homologous Protein Isoform 2 (CHP2), Na+/H+ Exchangers-Binding Protein, Is Expressed in Intestinal Epithelium.

“…Expression and Purification of NHE and CHP Fusion Proteins Expression plasmids for MBP and GST fusion proteins were constructed using vector pMAL-cri (New England Biolabs) or pGEX-4T (Amersham Biosciences), following the PCR-based method. 28) For production of His6-tagged rCHP1 or rCHP2, the coding sequence of rCHP1 or rCHP2 was cloned into expression vector pET21a (Novagen).…”

Interaction of the δ and b Subunits Contributes to F1 and F0 Interaction in the Escherichia coli F1F0-ATPase

“…Here, we have found a specific interaction between the b and ␦ subunits with two approaches; a genetic approach with the yeast two-hybrid system (26,27) and a biochemical approach with overproduction, purification, and in vitro binding of the subunits (28). The results directly indicate that the interaction of b and ␦ has an important role for F 1 and F 0 interaction.…”

“…The amounts of protein recovered were 92.4 mg and 14.3 mg for the supernatant and membrane fractions, respectively, after high speed centrifugation. The supernatant fraction was subjected to glutathioneSepharose (Pharmacia Biotech Inc.) (2.0 ml) column chromatography as described previously (28). The fraction eluted with 10 mM glutathione contained 26.6 mg of protein in which the major band stained by Coomassie Brilliant Blue was shown to be GST-b cyt .…”

“…Overexpression of GST 1 Fusion Subunit and Its Purification-The expression plasmid pGEX-b cyt was introduced into E. coli BL21 (28). Transformed E. coli was cultured in 500 ml of M9ZB medium supplemented with 0.2% glucose with vigorous shaking at 37°C.…”

“…Purified GST-␦ and GST-⑀ were prepared as described previously (28). GST-␦ (7.0 mg), GST-⑀ (6.0 mg), and GST-b cyt (8.2 mg) were digested with thrombin (11.5 units for 1 mg of fusion protein, Sigma T3010) for 20 h. Subsequently, the digested materials were subjected to glutathione-Sepharose affinity chromatography, and 2.2, 2.0, and 2.7 mg of ␦, ⑀, and b cyt , respectively, were obtained as practically homogeneous subunits.…”