title: A novel bacterial expression method with optimized parameters for very high yield production of triple-labeled proteins.
abstract: The Gram-negative bacterium Escherichia coli offer a means for rapid, high-yield, and economical production of recombinant proteins. However, when preparing protein samples for NMR, high-level production of functional isotopically labeled proteins can be quite challenging. This is especially true for the preparation of triple-labeled protein samples in D(2)O ((2)H/(13)C/(15)N). The large expense and time-consuming nature of triple-labeled protein production for NMR led us to revisit the current bacterial protein expression protocols. Our goal was to develop an efficient bacterial expression method for very high-level production of triple-labeled proteins that could be routinely utilized in every NMR lab without changing expression vectors or requiring fermentation. We developed a novel high cell-density IPTG-induction bacterial expression method that combines tightly controlled traditional IPTG-induction expression with the high cell-density of auto-induction expression. In addition, we optimize several key experimental protocols and parameters to ensure that our new high cell-density bacterial expression method routinely produces 14-25 mg of triple-labeled proteins and 15-35 mg of unlabeled proteins from 50-mL bacterial cell cultures.
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, MI, USA.
authors.forename: Victoria
authors.initials: V
authors.lastname: Murray
authors.forename: Yuefei
authors.initials: Y
authors.lastname: Huang
authors.forename: Jianglei
authors.initials: J
authors.lastname: Chen
authors.forename: Jianjun
authors.initials: J
authors.lastname: Wang
authors.forename: Qianqian
authors.initials: Q
authors.lastname: Li
mesh_terms: D019149:Bioreactors; D002247:Carbon Isotopes; D003903:Deuterium; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D007544:Isopropyl Thiogalactoside; D007553:Isotope Labeling / Q000379:methods*; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; D003903:Deuterium