Isolation of Escherichia coli inner membranes by metal affinity two-phase partitioning

Everberg, Henrik; Clough, Joanne; Henderson, Peter J. F.; Jergil, Bengt; Tjerneld, Folke; Ramírez, Irene Barinaga-Rementeria

doi:10.1016/j.chroma.2006.03.123

Cited by 37 publications

(22 citation statements)

References 40 publications

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“…Bacterial debris were removed by centrifugation at 10,000 × g for 10 min. The ISO membrane vesicles were further purified by metal affinity two-phase partitioning according to Everberg et al (25). Purification of ISO vesicles via a two-phase system in combination with Ni 2+ -NTA agarose resin was possible, as the His tag is cytoplasmatically located and, therefore, exposed in ISO vesicles.…”

Section: Methodsmentioning

confidence: 99%

Transport Mechanism and pH Regulation of the Na+/H+ Antiporter NhaA from Escherichia coli

Mager

Rimon

Padan

et al. 2011

Journal of Biological Chemistry

156

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Using an electrophysiological assay the activity of NhaA was tested in a wide pH range from pH 5.0 to 9.5. Forward and reverse transport directions were investigated at zero membrane potential using preparations with inside-out and right side-out-oriented transporters with Na+ or H+ gradients as the driving force. Under symmetrical pH conditions with a Na+ gradient for activation, both the wt and the pH-shifted G338S variant exhibit highly symmetrical transport activity with bell-shaped pH dependences, but the optimal pH was shifted 1.8 pH units to the acidic range in the variant. In both strains the pH dependence was associated with a systematic increase of the Km for Na+ at acidic pH. Under symmetrical Na+ concentration with a pH gradient for NhaA activation, an unexpected novel characteristic of the antiporter was revealed; rather than being down-regulated, it remained active even at pH as low as 5. These data allowed a transport mechanism to advance based on competing Na+ and H+ binding to a common transport site and a kinetic model to develop quantitatively explaining the experimental results. In support of these results, both alkaline pH and Na+ induced the conformational change of NhaA associated with NhaA cation translocation as demonstrated here by trypsin digestion. Furthermore, Na+ translocation was found to be associated with the displacement of a negative charge. In conclusion, the electrophysiological assay allows the revelation of the mechanism of NhaA antiport and sheds new light on the concept of NhaA pH regulation.

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Section: Methodsmentioning

confidence: 99%

Transport Mechanism and pH Regulation of the Na+/H+ Antiporter NhaA from Escherichia coli

Mager

Rimon

Padan

et al. 2011

Journal of Biological Chemistry

156

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“…The ISO membrane vesicles were further purified by metal affinity two-phase partitioning according to Ref. 21.…”

Section: Methodsmentioning

confidence: 99%

Differential Effects of Mutations on the Transport Properties of the Na+/H+ Antiporter NhaA from Escherichia coli

Mager

Braner

Kubsch

et al. 2013

Journal of Biological Chemistry

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Background: NhaA of Escherichia coli can serve as an example for pH regulation and transport mechanism in Na+/H+ exchangers.Results: Different mutations alter Na+ and/or H+ binding or conformational dynamics.Conclusion: Mutations in different segments of the protein change its pH profile by affecting different aspects of its transport mechanism.Significance: Single site mutations and kinetic analysis reveal determinants of Na+/H+ exchanger mechanism.

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“…His-tagged membrane proteins can then partition into the polymer phase (instead of the detergent-rich phase), allowing for a separation of tagged from untagged membrane proteins and the protection of the tagged protein from contact with extreme detergent concentrations (71). This method can also be used to purify membrane vesicles containing Histagged protein (72). …”

Section: How To Adapt the Protocol To Your Needsmentioning

confidence: 99%

Phase Separation in the Isolation and Purification of Membrane Proteins

Arnold

Linke

2007

BioTechniques

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Phase separation is a simple, efficient, and cheap method to purify and concentrate detergent-solubilized membrane proteins. In spite of this, phase separation is not widely used or even known among membrane protein scientists, and ready-to-use protocols are available for only relatively few detergent/membrane protein combinations. Here, we summarize the physical and chemical parameters that influence the phase separation behavior of detergents commonly used for membrane protein studies. Examples for the successful purification of membrane proteins using this method with different classes of detergents are provided. As the choice of the detergent is critical in many downstream applications (e.g., membrane protein crystallization or functional assays), we discuss how new phase separation protocols can be developed for a given detergent buffer system.

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Isolation of Escherichia coli inner membranes by metal affinity two-phase partitioning

Cited by 37 publications

References 40 publications

Transport Mechanism and pH Regulation of the Na+/H+ Antiporter NhaA from Escherichia coli

Transport Mechanism and pH Regulation of the Na+/H+ Antiporter NhaA from Escherichia coli

Differential Effects of Mutations on the Transport Properties of the Na+/H+ Antiporter NhaA from Escherichia coli

Phase Separation in the Isolation and Purification of Membrane Proteins

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