Protein expression and isotopic enrichment based on induction of the Entner-Doudoroff pathway in Escherichia coli.

School of Chemistry, Raymond and Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Ramat Aviv 69978, Tel Aviv, Israel.

Biochemical and biophysical research communications 2012 Oct 12

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The Entner-Doudoroff pathway is known to exist in many organisms including bacteria, archea and eukarya. Although the common route for carbon catabolism in Escherichia coli is the Embden-Meyerhof-Parnas pathway, it was shown that gluconate catabolism in E. coli occurs via the Entner-Doudoroff pathway. We demonstrate here that by supplying BL21(DE3) competent E.coli cells with gluconate in a minimal growth medium, protein expression can be induced. Nuclear magnetic resonance data of over-expressed ubiquitin show that by using [1-(13)C]-gluconate as the only carbon source, and (15)N-enriched ammonium chloride, sparse isotopic enrichment in the form of a spin-pair carbonyl-amide backbone enrichment is obtained. The specific amino acid labeling pattern is analyzed and is shown to be compatible with Entner-Doudoroff metabolism. Isotopic enrichment serves as a key factor in the biophysical characterization of proteins by various methods including nuclear magnetic resonance, mass spectrometry, infrared spectroscopy and more. Therefore, the method presented here can be applied to study proteins by obtaining sparse enrichment schemes that are not based on the regular glycolytic pathway, or to study the Entner-Doudoroff metabolism during protein expression. Copyright © 2012 Elsevier Inc. All rights reserved.

Citation

Bosmat Refaeli, Amir Goldbourt. Protein expression and isotopic enrichment based on induction of the Entner-Doudoroff pathway in Escherichia coli. Biochemical and biophysical research communications. 2012 Oct 12;427(1):154-8