An efficient and cost-effective isotope labeling protocol for proteins expressed in Escherichia coli (original) (raw)
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A rapid and robust method for selective isotope labeling of proteins
Methods
Amino-acid selective isotope labeling of proteins offers numerous advantages in mechanistic studies by revealing structural and functional information unattainable from a crystallographic approach. However, efficient labeling of proteins with selected amino acids necessitates auxotrophic hosts, which are often not available. We have constructed a set of auxotrophs in a commonly used Escherichia coli expression strain C43(DE3), a derivative of E. coli BL21(DE3), which can be used for isotopic labeling of individual amino acids or sets of amino acids. These strains have general applicability to either soluble or membrane proteins that can be expressed in E. coli. We present examples in which proteins are selectively labeled with 13 C-and 15 N-amino acids and studied using magic-angle spinning solid-state NMR and pulsed EPR, demonstrating the utility of these strains for biophysical characterization of membrane proteins, radical-generating enzymes and metalloproteins.
One-step amino acid selective isotope labeling of proteins in prototrophic Escherichia coli strains
Analytical Biochemistry, 2012
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 labeling 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.
A method for efficient isotopic labeling of recombinant proteins
Journal of Biomolecular Nmr, 2001
A rapid and efficient approach for preparing isotopically labeled recombinant proteins is presented. The method is demonstrated for 13C labeling of the C-terminal domain of angiopoietin-2, 15N labeling of ubiquitin and for 2H/13C/15N labeling of the Escherichia coli outer-membrane lipoprotein Lpp-56. The production method generates cell mass using unlabeled rich media followed by exchange into a small volume of labeled
Journal of Biomolecular NMR, 2013
The 1 H-13 C HMQC signals of the 13 CH 3 moieties of Ile, Leu, and Val residues, in an otherwise deuterated background, exhibit narrow line-widths, and thus are useful for investigating the structures and dynamics of larger proteins. This approach, named methyl TROSY, is economical as compared to laborious methods using chemically synthesized site-and stereo-specifically isotope-labeled amino acids, such as stereo-array isotope labeling amino acids, since moderately priced, commercially available isotope-labeled a-keto acid precursors can be used to prepare the necessary protein samples. The Ile d 1-methyls can be selectively labeled, using isotopelabeled a-ketobutyrates as precursors. However, it is still difficult to prepare a residue-selectively Leu and Val labeled protein, since these residues share a common biosynthetic intermediate, a-ketoisovalerate. Another hindering drawback in using the a-ketoisovalerate precursor is the lack of stereo-selectivity for Leu and Val methyls. Here we present a differential labeling method for Leu and Val residues, using four kinds of stereo-specifically 13 CH 3-labeled [U-2 H; 15 N]-leucine and-valine, which can be efficiently incorporated into a protein using Escherichia coli cellular expression. The method allows the differential labeling of Leu and Val residues with any combination of stereo-specifically isotope-labeled prochiral methyls. Since relatively small amounts of labeled leucine and valine are required to prepare the NMR samples; i.e., 2 and 10 mg/ 100 mL of culture for leucine and valine, respectively, with sufficient isotope incorporation efficiency, this approach will be a good alternative to the precursor methods. The feasibility of the method is demonstrated for 82 kDa malate synthase G.
mAbs, 2018
The widespread use of monoclonal antibodies (mAbs) as a platform for therapeutic drug development in the pharmaceutical industry has led to an increased interest in robust experimental approaches for assessment of mAb structure, stability and dynamics. The ability to enrich proteins with stable isotopes is a prerequisite for the in-depth application of many structural and biophysical methods, including nuclear magnetic resonance (NMR), small angle neutron scattering, neutron reflectometry, and quantitative mass spectrometry. While mAbs can typically be produced with very high yields using mammalian cell expression, stable isotope labeling using cell culture is expensive and often impractical. The most common and cost-efficient approach to label proteins is to express proteins in Escherichia coli grown in minimal media; however, such methods for mAbs have not been reported to date. Here we present, for the first time, the expression and purification of a stable isotope labeled mAb from a genetically engineered E. coli strain capable of forming disulfide bonds in its cytoplasm. It is shown using twodimensional NMR spectral fingerprinting that the unlabeled mAb and the mAb singly or triply labeled with 13 C, 15 N, 2 H are well folded, with only minor structural differences relative to the mammalian cellproduced mAb that are attributed to the lack of glycosylation in the Fc domain. This advancement of an E. coli-based mAb expression platform will facilitate the production of mAbs for in-depth structural characterization, including the high resolution investigation of mechanisms of action.
Journal of Biotechnology, 2004
A widely applicable cultivation strategy, which reduces the costs of expensive isotopes, is designed for maximal (98-100%) incorporation of [ 13 C] and [ 15 N] into labelled recombinant protein expressed in Escherichia coli, allowing better assignment of the resonances for NMR studies. Isotope labelling of the culture was performed throughout the complete process, starting from preculture. Sufficient biomass is first generated in a batch phase. Upon consumption of glucose, identified by a sharp drop of on-line monitored oxygen consumption, expression is induced and cultivation is continued under glucose-limited conditions as fed-batch process. Thereby a quantitative utilisation of the most expensive component [ 13 C]-glucose is achieved, while the approximate amount of the [ 15 N]-ammonium chloride to be incorporated is calculated from the scheduled biomass. The usefulness of the strategy is demonstrated with production of uniformly [ 13 C/ 15 N]-labelled tryparedoxin of Crithidia fasciculata. Ideal isotope incorporation and product quality is documented by MALDI-TOF mass spectrometry and two-and three-dimensional NMR spectra.
The15N isotope effect inEscherichia coli: A neutron can make the difference
PROTEOMICS, 2012
Several techniques based on stable isotope labeling are used for quantitative MS. These include stable isotope metabolic labeling methods for cells in culture as well as live organisms with the assumption that the stable isotope has no effect on the proteome. Here, we investigate the 15 N isotope effect on Escherichia coli cultures that were grown in either unlabeled (14 N) or 15 N-labeled media by LC-ESI-MS/MS-based relative protein quantification. Consistent protein expression level differences and altered growth rates were observed between 14 N and 15 N-labeled cultures. Furthermore, targeted metabolite analyses revealed altered metabolite levels between 14 N and 15 N-labeled bacteria. Our data demonstrate for the first time that the introduction of the 15 N isotope affects protein and metabolite levels in E. coli and underline the importance of implementing controls for unbiased protein quantification using stable isotope labeling techniques.
Affordable uniform isotope labeling with (2)H, (13)C and (15)N in insect cells
Journal of biomolecular NMR, 2015
For a wide range of proteins of high interest, the major obstacle for NMR studies is the lack of an affordable eukaryotic expression system for isotope labeling. Here, a simple and affordable protocol is presented to produce uniform labeled proteins in the most prevalent eukaryotic expression system for structural biology, namely Spodoptera frugiperda insect cells. Incorporation levels of 80 % can be achieved for (15)N and (13)C with yields comparable to expression in full media. For (2)H,(15)N and (2)H,(13)C,(15)N labeling, incorporation is only slightly lower with 75 and 73 %, respectively, and yields are typically twofold reduced. The media were optimized for isotope incorporation, reproducibility, simplicity and cost. High isotope incorporation levels for all labeling patterns are achieved by using labeled algal amino acid extracts and exploiting well-known biochemical pathways. The final formulation consists of just five commercially available components, at costs 12-fold lower...
Preparation of amino-acid-type selective isotope labeling of protein expressed in Pichia pastoris
Proteins: Structure, Function, and Bioinformatics, 2005
We report the culture conditions for successful amino-acid-type selective (AATS) isotope labeling of protein expressed in Pichia pastoris (P. pastoris). Rhodostomin (Rho), a six disulfidebonded protein expressed in P. pastoris with the correct fold, was used to optimize the culture conditions. The concentrations of [␣-15 N] selective amino acid, nonlabeled amino acids, and ammonium chloride, as well as induction time, were optimized to avoid scrambling and to increase the incorporation rate and protein yield. The optimized protocol was successfully applied to produce AATS isotopelabeled Rho. The labeling of [␣-15 N]Cys has a 50% incorporation rate, and all 12 cysteine resonances were observed in HSQC spectrum. The labeling of [␣-15 N]Leu,-Lys, and-Met amino acids has an incorporation rate greater than 65%, and the expected number of resonances in the HSQC spectra were observed. In contrast, the labeling of [␣-15 N]Asp and-Gly amino acids has a low incorporation rate and the scrambling problem. In addition, the culture condition was successfully applied to label dendroaspin (Den), a four disulfide-bonded protein expressed in P. pastoris. Therefore, the described condition should be generally applicable to other proteins produced in the P. pastoris expression system. This is the first report to present a protocol for AATS isotope labeling of protein expressed in P.