Auto-induction medium for the production of [U-15N]- and [U-13C, U-15N]-labeled proteins for NMR screening and structure determination (original) (raw)
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Journal of Structural and Functional Genomics, 2000
The use of 2-L polyethylene terephthalate beverage bottles as a bacterial culture vessel has been recently introduced as an enabling technology for high-throughput structural biology [Sanville Millard, C. et al., 2003. Protein Express. Purif. 29, 311-320]. In the article following this one [Stols et al., this issue,, this approach was elaborated for selenomethionine labeling used for multiwavelength anomalous dispersion phasing in the Xray crystallographic determinations of protein structure. Herein, we report an effective and reproducible schedule for uniform 15 N-and 13 C-labeling of recombinant proteins in 2-L beverage bottles for structural determination by NMR spectroscopy. As an example, three target proteins selected from Arabidopsis thaliana were expressed in Escherichia coli Rosetta (DE3)/pLysS from a T7-based expression vector, purified, and characterized by electrospray ionization mass spectrometry and NMR analysis by 1 H-15 N heteronuclear single quantum correlation spectroscopy. The results show that expressions in the unlabeled medium provide a suitable control for estimation of the level of production of the labeled protein. Mass spectral characterizations show that the purified proteins contained a level of isotopic incorporation equivalent to the isotopically labeled materials initially present in the growth medium, while NMR analysis of the [U-15 N]-labeled proteins provided a convenient method to assess the solution state properties of the target protein prior to production of a more costly double-labeled sample.
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.
Journal of Biomolecular Nmr, 1999
A selective protonation strategy is described that uses [3-2 H] 13 C α-ketoisovalerate to introduce (1 H-δ methyl)leucine and (1 H-γ methyl)-valine into 15 N-, 13 C-, 2 H-labeled proteins. A minimum level of 90% incorporation of label into both leucine and valine methyl groups is obtained by inclusion of ≈100 mg/L α-ketoisovalerate in the bacterial growth medium. Addition of [3,3-2 H 2 ] α-ketobutyrate to the expression media (D 2 O solvent) results in the production of proteins with (1 H-δ1 methyl)-isoleucine (>90% incorporation). 1 H-13 C HSQC correlation spectroscopy establishes that CH 2 D and CHD 2 isotopomers are not produced with this method. This approach offers enhanced labeling of Leu methyl groups over previous methods that utilize Val as the labeling agent and is more cost effective.
Journal of biomolecular NMR, 1999
A selective protonation strategy is described that uses [3-2H] 13C alpha-ketoisovalerate to introduce (1H-delta methyl)-leucine and (1H-gamma methyl)-valine into 15N-, 13C-, 2H-labeled proteins. A minimum level of 90% incorporation of label into both leucine and valine methyl groups is obtained by inclusion of approximately 100 mg/L alpha-ketoisovalerate in the bacterial growth medium. Addition of [3,3-2H2] alpha-ketobutyrate to the expression media (D2O solvent) results in the production of proteins with (1H-delta1 methyl)-isoleucine (> 90% incorporation). 1H-13C HSQC correlation spectroscopy establishes that CH2D and CHD2 isotopomers are not produced with this method. This approach offers enhanced labeling of Leu methyl groups over previous methods that utilize Val as the labeling agent and is more cost effective.
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.
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.
Protein Expression and Purification, 1997
active nuclei. Protein may be globally labeled by enrich-Methods for the efficient use of the 13 C-labeled nutri-ment with 13 C and/or 15 N at all positions and specifically ents, glucose and histidine, in the production of recom-labeled at amino acid residues or even certain atoms binant protein were developed to provide the large within these residues. Although media mixes enriched amount of sample required for NMR studies. The nutriwith 13 C and 15 N are commercially available and may ent requirements were reduced by determining the be used to produce globally labeled samples, a minimal minimum amount of these metabolites needed during medium is required for specific labeling because it allows both the growth and the induction phases of the control of all nutrients included in the broth. However, a BL21(DE3) and newly constructed BL21(DE3) histitypical minimal medium (M9) cannot support the same dine auxotrophic Escherichia coli cultures. These level of growth as a richer medium and thus limits the methods were developed using the separate bisphosamount of protein produced by limiting the cell density phatase domain of rat liver 6-phosphofructo-2-kinase/ of the culture. A fortified minimal medium (M10) that fructose-2,6-bisphosphatase, which is expressed to can support both bacterial growth as well as LB and high levels in the pET3a/BL21(DE3) bacterial system. provide for labeling has been described previously (3). Use of the optimized expression methods reduced the Because labeling with 13 C metabolites is very expensive, requirements for the labeled nutrients, glucose and it is advantageous to minimize the amount of label rehistidine, by 90 and 93.8%, respectively. The savings quired for production of the POI. realized by use of the minimized media and modified As a model POI we used the separately expressed induction protocols were obtained without significant bisphosphatase domain (ND249) of the bifunctional enreduction of the yield of purified protein. Comprehenzyme rat liver 6-phosphofructo-2-kinase/fructose-2,6-79
Preparation of site-specifically labeled fluorinated proteins for 19F-NMR structural characterization
A straightforward protocol for the site-specific incorporation of a 19 F label into any protein in vivo is described. This is done using a plasmid containing an orthogonal aminoacyl-tRNA synthetase/tRNA CUA that incorporates L-4-trifluoromethylphenylalanine in response to the amber codon UAG. This method improves on other in vivo methods because the 19 F label is incorporated into only one location on the protein of interest and that protein can easily be produced in large quantities at low cost. The protocol for producing 19 F-labeled protein is similar to expressing protein in Escherichia coli and takes 4 d to obtain pure protein starting from the appropriate vectors.
Chemical synthesis of proteins: a tool for protein labeling
2010
The need for protein modification strategies pag. Chemical synthesis of protein pag. Classical organic synthesis in solution pag. Solid phase peptide synthesis pag. Chemical ligation reaction pag. Chemical ligation of unprotected peptide segments pag. Chemical ligation reactions yielding a non-native link pag. Native Chemical Ligation pag. Expressed Protein Ligation pag. Performing Native Chemical Ligation and Expressed Protein Ligation pag. Production of thioester peptides pag. Production of N-terminal Cys peptide pag. Ligation of multiple peptide fragments pag. Protein semisynthesis by trans-splicing pag. Applications of NCL and EPL pag. Introduction of fluorescent probes pag. Introduction of isotopic probes pag. Introduction of post-translational modifications: phosphorylation, glycosylation, lipidation, ubiquitylation pag. EXPERIMENTAL SECTION pag. Materials and Instruments pag. Methods pag. Antibiotics pag. Solid and liquid media for bacterial strains pag. Preparation of E. coli TOP F'10 competent cells and transformation by electroporation pag. Preparation of E. coli competent cells and transformation by heat shock pag. Electrophoretic analysis of DNA pag. Electrophoretic analysis of proteins (SDS-PAGE) pag. Determination of the protein concentration pag. Bioinformatic tools pag. Cloning procedure pag. Purification pag. First labeling reaction pag. Native Chemical Ligation with L-Cys pag. Second labeling reaction pag. Mono-labeled and unlabeled control constructs preparation pag. Spectroscopic characterization of CTPR3 protein variants pag. CD characterization pag. Fluorescence anisotropy measurements pag. Ensemble-FRET studies on Doubly-labeled CTPR3 protein variants pag. Chemical denaturation studies by CD and FRET pag. CONCLUSION pag. ABBREVIATIONS Acm acetamidomethyl AW azatryptophan BAL backbone amide linker Boc t-butoxycarbonyl BSA bovine serum albumin c-Abl Abelson nonreceptor protein tyrosine kinase CD circular dicroism ctpr3 Consensus Tetratrico Peptide Repeat protein 3 gene CTPR3 Consensus Tetratrico Peptide Repeat protein 3 Dab dabcyl Dansyl 5-(dimethylamino)-naphtalene-sulfonamide DBU 1,8-diazabicyclo[5.4.0]undec-7-ene Dbz diaminobenzoic acid DCM dichloromethane DIPEA N,N-diisopropylethylamine DMAP 4-Dimethylaminopyridine DMF dimethylformamide DMSO dimethylsulfoxide DNA deoxyribonucleic acid dNTP deoxy-nucleotide tri-phosphate ε extinction coefficient E FRET efficiency E. coli Escherichia coli EDT ethandithiol EDTA ethylene-diamino-tetraacetic acid EPL Expressed Protein Ligation ESI electron spray ionization source FCS Fluorescence Correlation Spectroscopy Fmoc fluorenylmethyloxycarbonyl chloride FP Fluorescence Polarization FPLC Fast Protein Liquid Chromatography FRET Förster Resonance Energy Transfer GdnHCl guanidinium hydrochloride GFP Green Fluorescent Protein Gla PDA photo diode array pI isoelectric point
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.