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

Journal of Chromatography. a
Henrik EverbergIrene Barinaga-Rementeria Ramírez

Abstract

As reduction of sample complexity is a central issue in membrane proteomic research, the need for new pre-fractionation methods is significant. Here we present a method for fast and efficient enrichment of Escherichia coli inner membranes expressing a His-tagged integral membrane L-fucose-proton symporter (FucP). An enriched inner membrane fraction was obtained from a crude membrane mixture using affinity two-phase partitioning in combination with nickel-nitrilotriacetic acid (Ni-NTA) immobilized on agarose beads. Due to interaction between the beads and FucP, inner membranes were selectively partitioned to the bottom phase of a polymer/polymer aqueous two-phase system consisting of poly(ethylene glycol) (PEG) and dextran. The partitioning of membranes was monitored by assaying the activity of an inner membrane marker protein and measuring the total protein content in both phases. The enrichment of inner membrane proteins in the dextran phase was also investigated by proteomic methodology, including sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), trypsin digestion and liquid chromatography in combination with tandem mass spectrometry (LC-MS/MS). Using a high level of significance (99.95%) in the subsequent...Continue Reading

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Citations

Oct 27, 2012·Applied Microbiology and Biotechnology·Randy Chi Fai CheungTzi Bun Ng
Mar 14, 2012·Proceedings of the National Academy of Sciences of the United States of America·Gary J Pielak, Fang Tian
Jul 10, 2013·The Journal of Biological Chemistry·Thomas MagerKlaus Fendler
Jan 22, 2013·Annual Review of Physical Chemistry·Chi-Yuan Cheng, Songi Han
Jan 30, 2013·Journal of Chromatography. a·Guilherme Dias RodriguesMaria C Hespanhol da Silva
May 25, 2012·Journal of Chromatography. a·Federico Ruiz-RuizMarco Rito-Palomares
May 15, 2015·Protein Science : a Publication of the Protein Society·Leonid S Brown, Vladimir Ladizhansky
Nov 6, 2007·Progress in Lipid Research·Claude Wolf, Peter J Quinn
Mar 8, 2008·Proteomics·Nikhil PatelSanjoy K Bhattacharya
Oct 12, 2010·Mass Spectrometry Reviews·Maroun Bou KhalilDaniel Figeys
Apr 12, 2015·Biophysical Journal·Meaghan E WardVladimir Ladizhansky
Apr 13, 2015·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Tatsuya OshimaYoshinari Baba
Nov 21, 2007·BioTechniques·Thomas Arnold, Dirk Linke

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