title: Protein expression and isotopic enrichment based on induction of the Entner-Doudoroff pathway in Escherichia coli.
abstract: 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.
authors.affiliation: School of Chemistry, Raymond and Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Ramat Aviv 69978, Tel Aviv, Israel.
authors.forename: Bosmat
authors.initials: B
authors.lastname: Refaeli
authors.forename: Amir
authors.initials: A
authors.lastname: Goldbourt
mesh_terms: D000596:Amino Acids / Q000032:analysis / Q000378:metabolism; D002247:Carbon Isotopes / Q000378:metabolism; D003470:Culture Media / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000737:chemistry; D005942:Gluconates / Q000378:metabolism*; D007553:Isotope Labeling / Q000379:methods*; D013058:Mass Spectrometry; D053858:Metabolic Networks and Pathways; D019906:Nuclear Magnetic Resonance, Biomolecular; D014176:Protein Biosynthesis; D011549:Pseudomonas / Q000378:metabolism; D013055:Spectrophotometry, Infrared; D025801:Ubiquitin / Q000378:metabolism
chemical_list: D000596:Amino Acids; D002247:Carbon Isotopes; D003470:Culture Media; D029968:Escherichia coli Proteins; D005942:Gluconates; D025801:Ubiquitin