Notes & Tips One-step amino acid selective isotope labeling of proteins in prototrophic Escherichia coli strains Christopher O’Grady, Benjamin L. Rempel, Akosiererem Sokaribo, Sergiy Nokhrin, Oleg Y. Dmitriev ⇑ Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5E5 article info Article history: Received 2 February 2012 Received in revised form 10 April 2012 Accepted 13 April 2012 Available online 23 April 2012 Keywords: Selective isotope labeling Protein NMR abstract Amino acid selective isotope labeling is a useful approach to simplification of nuclear magnetic resonance (NMR) spectra of large proteins. Cell-free protein synthesis offers essentially unlimited flexibility of label- ing patterns but is labor-intensive and expensive. In vivo labeling is simple in principle but generally requires auxotrophic strains, inhibitors of amino acid synthesis, or complex media formulations. We describe a simple procedure for amino acid selective labeling of proteins expressed in prototrophic Escherichia coli strains. Excellent labeling selectivity was achieved for histidine, lysine, methionine, and alanine. Simplicity and robustness of this protocol make it a useful tool for protein NMR. Ó 2012 Elsevier Inc. All rights reserved. Nuclear magnetic resonance (NMR) 1 spectra of large proteins suffer from peak overlap and chemical shift degeneracy that compli- cate the chemical shift assignment process. Even for medium-sized proteins, spectral overlap in two-dimensional spectra, such as a 1 H, 15 N-HSQC (heteronuclear single quantum coherence), can be se- vere enough to hamper ligand binding studies by chemical shift per- turbation analysis. Amino acid selective isotope labeling is widely used to simplify NMR spectra by eliminating signals from unlabeled amino acid residues. Selective labeling in cell-free protein systems [1–3], also known as in vitro translation, offers essentially unlimited flexibility of labeling patterns but is labor-intensive and expensive. In contrast, in vivo labeling is simple in principle, but its flexibility and selectivity are limited. In general, for in vivo labeling the protein of interest is expressed in Escherichia coli, or (less commonly) other bacterial [4] or yeast cells, while isotopically labeled amino acids are added to the growth medium [5]. An interesting and rather cost-effective variation of the in vivo labeling approach is the selec- tive unlabeling or inverse labeling technique, where one or several natural isotopic abundance amino acids are introduced into the otherwise uniformly 15 N- or 13 C, 15 N-labeled protein to assist with backbone chemical shift assignment by the exclusion method [6,7]. Three techniques are commonly used to prevent isotope dilution and label scrambling due to metabolic reactions in the cell that pro- duce unlabeled amino acids, on the one hand, and convert the externally added isotopically labeled amino acids to the different ones, on the other. Use of an auxotrophic strain that is deficient in the synthesis of a given amino acid prevents isotope dilution that results from incorporation of the newly synthesized unlabeled ami- no acid into the expressed protein [8,9]. However, constructing strains with multiple auxotrophic markers is impractical, and such strains usually have poor viability. Alternatively, specific inhibitors of amino acid synthesis pathways can be used to suppress produc- tion of the chosen amino acids in the cell [10]. However, the arsenal and specificity of such inhibitors are limited. Except for the amino acids that are terminal products of anabolic pathways, such as argi- nine, glycine, cysteine, histidine, lysine, and methionine, additional measures are required to prevent isotope scrambling that degrades labeling selectivity. Commonly, this is achieved by supplementing the minimal growth medium with all of the amino acids in the unla- beled form except for the one chosen for selective labeling. None of these methods offers a generally applicable approach to simulta- neously achieving high selectivity and high labeling efficiency. Sev- eral methods employ metabolic precursors to selectively direct isotopic label incorporation into the specific groups in the amino acid side chains [11–13], but these techniques are applicable to only a few particular amino acids and do not allow selective label- ing of backbone amides. Despite obvious limitations, amino acid selective labeling in vivo combined with modern heteronuclear multidimensional NMR experiments remains a useful tool for resolving chemical shift assignment ambiguities and simplifying NMR spectra for ligand binding studies. In this article, we show that highly efficient and extremely simple selective labeling with some amino acids can be achieved in the common prototrophic E. coli strains widely used for the expression of recombinant proteins. We used the 165-amino-acid N domain of Wilson disease protein (ATP7B) for testing amino acid selective labeling in the 0003-2697/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ab.2012.04.019 ⇑ Corresponding author. Address: Department of Biochemistry, University of Saskatchewan, 107 Wiggins Rd., Saskatoon, Canada SK S7N 5E5. Fax: +1 306 966 4390. E-mail address: oleg.dmitriev@usask.ca (O.Y. Dmitriev). 1 Abbreviations used: NMR, nuclear magnetic resonance; HSQC, heteronuclear single quantum coherence. Analytical Biochemistry 426 (2012) 126–128 Contents lists available at SciVerse ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio