One-step amino acid selective isotope labeling of proteins in prototrophic Escherichia coli strains (original) (raw)
Related papers
2001
Nuclear magnetic resonance is an important tool for high-resolution structural studies of proteins. It demands high protein concentration and high purity; however, the expression of proteins at high levels often leads to protein aggregation and the protein purification step can correspond to a high percentage of the overall time in the structural determination process. In the present article we show that the step of sample optimization can be simplified by selective labeling the heterologous protein expressed in Escherichia coli by the use of rifampicin. Yeast thioredoxin and a coix transcription factor Opaque 2 leucine zipper (LZ) were used to show the effectiveness of the protocol. The 1 H/ 15 N heteronuclear correlation two-dimensional NMR spectrum (HMQC) of the selective 15 N-labeled thioredoxin without any purification is remarkably similar to the spectrum of the purified protein. The method has high yields and a good 1 H/ 15 N HMQC spectrum can be obtained with 50 ml of M9 growth medium. Opaque 2 LZ, a difficult protein due to the lower expression level and high hydrophobicity, was also probed. The 15 N-edited spectrum of Opaque 2 LZ showed only the resonances of the protein of heterologous expression (Opaque 2 LZ) while the 1 H spectrum shows several other resonances from other proteins of the cell lysate. The demand for a fast methodology for structural determination is increasing with the advent of genome/proteome projects. Selective labeling the heterologous protein can speed up NMR structural studies as well as NMR-based drug screening. This methodology is especially effective for difficult proteins such as hydrophobic transcription factors, membrane proteins, and others.
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.
2001
Nuclear magnetic resonance is an important tool for high-resolution structural studies of proteins. It demands high protein concentration and high purity; however, the expression of proteins at high levels often leads to protein aggregation and the protein purification step can correspond to a high percentage of the overall time in the structural determination process. In the present article we show that the step of sample optimization can be simplified by selective labeling the heterologous protein expressed in Escherichia coli by the use of rifampicin. Yeast thioredoxin and a coix transcription factor Opaque 2 leucine zipper (LZ) were used to show the effectiveness of the protocol. The 1 H/ 15 N heteronuclear correlation two-dimensional NMR spectrum (HMQC) of the selective 15 N-labeled thioredoxin without any purification is remarkably similar to the spectrum of the purified protein. The method has high yields and a good 1 H/ 15 N HMQC spectrum can be obtained with 50 ml of M9 growth medium. Opaque 2 LZ, a difficult protein due to the lower expression level and high hydrophobicity, was also probed. The 15 N-edited spectrum of Opaque 2 LZ showed only the resonances of the protein of heterologous expression (Opaque 2 LZ) while the 1 H spectrum shows several other resonances from other proteins of the cell lysate. The demand for a fast methodology for structural determination is increasing with the advent of genome/proteome projects. Selective labeling the heterologous protein can speed up NMR structural studies as well as NMR-based drug screening. This methodology is especially effective for difficult proteins such as hydrophobic transcription factors, membrane proteins, and others.
High yield expression of proteins in <i>E. coli</i> for NMR studies
Advances in Bioscience and Biotechnology, 2013
In recent years, high yield expression of proteins in E. coli has witnessed rapid progress with developments of new methodologies and technologies. An important advancement has been the development of novel recombinant cloning approaches and protocols to express heterologous proteins for Nuclear Magnetic Resonance (NMR) studies and for isotopic enrichment. Isotope labeling in NMR is necessary for rapid acquisition of high dimensional spectra for structural studies. In addition, higher yield of proteins using various solubility and affinity tags has made protein overexpression cost-effective. Taken together, these methods have opened new avenues for structural studies of proteins and their interactions. This article deals with the different techniques that are employed for over-expression of proteins in E. coli and different methods used for isotope labeling of proteins vis-à-vis NMR spectroscopy.
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.
Optimal isotope labelling for NMR protein structure determinations
Nature, 2006
Nuclear-magnetic-resonance spectroscopy can determine the three-dimensional structure of proteins in solution. However, its potential has been limited by the difficulty of interpreting NMR spectra in the presence of broadened and overlapping resonance lines and low signal-to-noise ratios. Here we present stereo-array isotope labelling (SAIL), a technique that can overcome many of these problems by applying a complete stereospecific and regiospecific pattern of stable isotopes that is optimal with regard to the quality and information content of the resulting NMR spectra. SAIL uses exclusively chemically and enzymatically synthesized amino acids for cell-free protein expression. We demonstrate for the 17-kDa protein calmodulin and the 41-kDa maltodextrin-binding protein that SAIL offers sharpened lines, spectral simplification without loss of information, and the ability to rapidly collect the structural restraints required to solve a high-quality solution structure for proteins twice as large as commonly solved by NMR. It thus makes a large class of proteins newly accessible to detailed solution structure determination.
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.
An efficient and cost-effective isotope labeling protocol for proteins expressed in Escherichia coli
Journal of biomolecular NMR, 1998
A cost-effective protocol for uniform 15N and/or 13C isotope labeling of bacterially expressed proteins is presented. Unlike most standard protocols, cells are initially grown in a medium containing nutrients at natural abundance and isotopically labeled nutrients are only supplied at the later stages of growth and during protein expression. This permits the accumulation of a large cell mass without the need to employ expensive isotopically labeled nutrients. The abrupt decrease in oxygen consumption that occurs upon complete exhaustion of essential nutrients is used to precisely time the switch between unlabeled and labeled nutrients. Application of the protocol is demonstrated for wild-type and a mutant of the N-terminal zinc-binding domain of HIV-1 integrase.
Chapter 2 Isotope-Aided Methods for Biological NMR Spectroscopy : Past , Present , and Future
2019
This chapter starts by providing a historical background of our research endeavors over the past half-century to develop various isotope-aided methods in biological NMR spectroscopy, since innovations bloom only on the rich ground cultivated by previous investigators. We then focused on the stereo-array isotope-labeling (SAIL) method, one of our recent accomplishments, which culminates the isotope-aided NMR technologies for structural studies of proteins from various aspects: accurate structural determinations of large proteins, elaboration for automated structural determination, highly efficient and versatile residue-selective methyl labeling with newly developed auxotrophic E. coli strains, large-amplitude slow-breathing motion (LASBM) as revealed by the aromatic ring flipping of the residues in ligand-binding interfaces, and applications of the deuterium-induced C-NMR isotope shift to investigate the hydrogen exchange phenomena of side-chain polar groups. Meanwhile, the expected ...