title: Efficient isotopic tryptophan labeling of membrane proteins by an indole controlled process conduct.
abstract: A protocol for the efficient isotopic labeling of large G protein-coupled receptors with tryptophan in Escherichia coli as expression host was developed that sufficiently suppressed the naturally occurring L-tryptophan indole lyase, which cleaves tryptophan into indole, pyruvate, and ammonia resulting in scrambling of the isotopic label in the protein. Indole produced by the tryptophanase is naturally used as messenger for cell-cell communication. Detailed analysis of different process conducts led to the optimal expression strategy, which mimicked cell-cell communication by the addition of indole during expression. Discrete concentrations of indole and (15) N2 -L-tryptophan at dedicated time points in the fermentation drastically increased the isotopic labeling efficiency. Isotope scrambling was only observed in glutamine, asparagine, and arginine side chains but not in the backbone. This strategy allows producing specifically tryptophan labeled membrane proteins at high concentrations avoiding the disadvantages of the often low yields of auxotrophic E. coli strains. In the fermentation process carried out according to this protocol, we produced ∼15 mg of tryptophan labeled neuropeptide Y receptor type 2 per liter medium.
authors.affiliation: Institute of Medical Physics and Biophysics, University of Leipzig, Härtelstr. 16-18, Leipzig 04107, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Berger
authors.forename: Sandra
authors.initials: S
authors.lastname: Berndt
authors.forename: Annelie
authors.initials: A
authors.lastname: Pichert
authors.forename: Stephan
authors.initials: S
authors.lastname: Theisgen
authors.forename: Daniel
authors.initials: D
authors.lastname: Huster
mesh_terms: D019149:Bioreactors; D001709:Biotechnology / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism; D005285:Fermentation; D006801:Humans; D007211:Indoles / Q000378:metabolism*; D007544:Isopropyl Thiogalactoside / Q000737:chemistry; D007553:Isotope Labeling / Q000379:methods*; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry* / Q000378:metabolism; D014368:Tryptophanase / Q000737:chemistry / Q000378:metabolism
chemical_list: D007211:Indoles; D009587:Nitrogen Isotopes; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; C030374:indole; D014364:Tryptophan; D014368:Tryptophanase