当前位置： SCI文献检索 > JOURNAL OF BIOMOLECULAR NMR期刊下所有文献 > A simple protocol for expression of isotope-labeled proteins in Escherichia coli grown in shaker flasks at high cell density.

A simple protocol for expression of isotope-labeled proteins in Escherichia coli grown in shaker flasks at high cell density.

Abstract：

:Protein expression in E. coli grown in shaker flasks is a routine and pivotal tool in many research laboratories. To maximize protein yields, cells are normally induced in the middle of the linear growth phase, typically at an OD600 of ≤ 1 for cells grown in Luria-Bertani (LB) medium at 37 °C. We recently showed that the E. coli linear growth phase can be extended to higher cell density when cells are cultured under less than optimal conditions such as in minimal medium and/or at lower temperatures. Maximizing the yield of protein per unit volume of culture is important for reducing the costs, especially when isotopically labeling is required. Here, we present a modified minimal medium and a simple protocol that can increase the protein yield up to fourfold in a pH-stabilized LB medium and up to sevenfold in a modified M9+ medium (M9++). When M9++ medium coupled with the high density (OD600 ~ 6) cell growth protocol are used to express uniformly 15N- or 15N/13C-labeled proteins, the amount of 15NH4Cl and 13C6-glucose for a given cell mass is reduced by 50% and ~ 65%, respectively, relative to the traditional low density (OD600 ~ 1) cell growth protocol with M9 medium; the inclusion of 0.1% LB in the minimal medium permits a reduction in the concentration of both the trace element solution and MgCl2, which can cause precipitation. Mass data indicate that inclusion of 0.1% LB does not significantly affect the isotope enrichment level.

journal_name

J Biomol NMR

journal_title

Journal of biomolecular NMR

authors

Cai M,Huang Y,Craigie R,Clore GM

doi

10.1007/s10858-019-00285-x

subject

Has Abstract

pub_date

2019-12-01 00:00:00

pages

743-748

issue

12

eissn

0925-2738

issn

1573-5001

pii

10.1007/s10858-019-00285-x

journal_volume

73

pub_type

杂志文章

相关文献

JOURNAL OF BIOMOLECULAR NMR文献大全
  • Isotope labeling of mammalian GPCRs in HEK293 cells and characterization of the C-terminus of bovine rhodopsin by high resolution liquid NMR spectroscopy.

    abstract：:High amino acid coverage labeling of the mammalian G protein coupled receptors (GPCR) rhodopsin was established with 15N and 15N/13C isotopes. Rhodopsin was expressed at preparative scale in HEK293S cells and studied in full-length by NMR spectroscopy in detergent micelle solution. This resulted in the assignment and ...

    journal_title：Journal of biomolecular NMR

    pub_type： 杂志文章

    doi：10.1007/s10858-007-9205-3

    authors： Werner K,Richter C,Klein-Seetharaman J,Schwalbe H

    更新日期：2008-01-01 00:00:00

  • Archiving and disseminating integrative structure models.

    abstract：:Limitations in the applicability, accuracy, and precision of individual structure characterization methods can sometimes be overcome via an integrative modeling approach that relies on information from all available sources, including all available experimental data and prior models. The open-source Integrative Modeli...

    journal_title：Journal of biomolecular NMR

    pub_type： 杂志文章

    doi：10.1007/s10858-019-00264-2

    authors： Vallat B,Webb B,Westbrook J,Sali A,Berman HM

    更新日期：2019-07-01 00:00:00

  • Determination of a complete set of coupling constants in 13C-labeled oligonucleotides.

    abstract：:Three experiments are introduced to determine a complete set of coupling constants in RNA oligomers. In the HCCH-E.COSY experiment, the vicinal proton-proton coupling constants can be measured with high accuracy. In the P-FIDS-CT-HSQC experiment, vicinal proton-phosphorus and carbon-phosphorus couplings are measured t...

    journal_title：Journal of biomolecular NMR

    pub_type： 杂志文章

    doi：10.1007/BF00404274

    authors： Schwalbe H,Marino JP,King GC,Wechselberger R,Bermel W,Griesinger C

    更新日期：1994-09-01 00:00:00

  • Automated robust and accurate assignment of protein resonances for solid state NMR.

    abstract：:The process of resonance assignment represents a time-consuming and potentially error-prone bottleneck in structural studies of proteins by solid-state NMR (ssNMR). Software for the automation of this process is therefore of high interest. Procedures developed through the last decades for solution-state NMR are not di...

    journal_title：Journal of biomolecular NMR

    pub_type： 杂志文章

    doi：10.1007/s10858-014-9835-1

    authors： Nielsen JT,Kulminskaya N,Bjerring M,Nielsen NC

    更新日期：2014-06-01 00:00:00

  • HIFI-C: a robust and fast method for determining NMR couplings from adaptive 3D to 2D projections.

    abstract：:We describe a novel method for the robust, rapid, and reliable determination of J couplings in multi-dimensional NMR coupling data, including small couplings from larger proteins. The method, "High-resolution Iterative Frequency Identification of Couplings" (HIFI-C) is an extension of the adaptive and intelligent data...

    journal_title：Journal of biomolecular NMR

    pub_type： 杂志文章

    doi：10.1007/s10858-007-9173-7

    authors： Cornilescu G,Bahrami A,Tonelli M,Markley JL,Eghbalnia HR

    更新日期：2007-08-01 00:00:00

  • Correlated motions of successive amide N-H bonds in proteins.

    abstract：:New nuclear magnetic resonance (NMR) methods are described for the measurement of cross-correlation rates of zero- and double-quantum coherences involving two nitrogen nuclei belonging to successive amino acids in proteins and peptides. Rates due to the concerted fluctuations of two NH(N) dipole-dipole interactions an...

    journal_title：Journal of biomolecular NMR

    pub_type： 杂志文章

    doi：10.1023/a:1023076212536

    authors： Pelupessy P,Ravindranathan S,Bodenhausen G

    更新日期：2003-04-01 00:00:00

  • 13C alpha-NMR assignments of melittin in methanol and chemical shift correlations with secondary structure.

    abstract：:Melittin is a naturally occurring hexacosa peptide which forms an amphiphilic helix in methanol, a random coil in water, and a tetramer of helices at basic pH or in the presence of a high salt concentration. The monomeric structure in methanol has been well characterized by proton NMR (Pastore et al. (1989) Eur. Bioph...

    journal_title：Journal of biomolecular NMR

    pub_type： 杂志文章

    doi：10.1007/BF00198369

    authors： Buckley P,Edison AS,Kemple MD,Prendergast FG

    更新日期：1993-11-01 00:00:00

  • Solid-state NMR, electrophysiology and molecular dynamics characterization of human VDAC2.

    abstract：:The voltage-dependent anion channel (VDAC) is the most abundant protein of the outer mitochondrial membrane and constitutes the major pathway for the transport of ADP, ATP, and other metabolites. In this multidisciplinary study we combined solid-state NMR, electrophysiology, and molecular dynamics simulations, to stud...

    journal_title：Journal of biomolecular NMR

    pub_type： 杂志文章

    doi：10.1007/s10858-014-9876-5

    authors： Gattin Z,Schneider R,Laukat Y,Giller K,Maier E,Zweckstetter M,Griesinger C,Benz R,Becker S,Lange A

    更新日期：2015-04-01 00:00:00

  • 'Random coil' 1H chemical shifts obtained as a function of temperature and trifluoroethanol concentration for the peptide series GGXGG.

    abstract：:Proton chemical shifts of a series of disordered linear peptides (H-Gly-Gly-X-Gly-Gly-OH, with X being one of the 20 naturally occurring amino acids) have been obtained using 1D and 2D 1H NMR at pH 5.0 as a function of temperature and solvent composition. The use of 2D methods has allowed some ambiguities in side-chai...

    journal_title：Journal of biomolecular NMR

    pub_type： 杂志文章

    doi：10.1007/BF00227466

    authors： Merutka G,Dyson HJ,Wright PE

    更新日期：1995-01-01 00:00:00

  • Protein resonance assignment at MAS frequencies approaching 100 kHz: a quantitative comparison of J-coupling and dipolar-coupling-based transfer methods.

    abstract：:We discuss the optimum experimental conditions to obtain assignment spectra for solid proteins at magic-angle spinning (MAS) frequencies around 100 kHz. We present a systematic examination of the MAS dependence of the amide proton T 2' times and a site-specific comparison of T 2' at 93 kHz versus 60 kHz MAS frequency....

    journal_title：Journal of biomolecular NMR

    pub_type： 杂志文章

    doi：10.1007/s10858-015-9975-y

    authors： Penzel S,Smith AA,Agarwal V,Hunkeler A,Org ML,Samoson A,Böckmann A,Ernst M,Meier BH

    更新日期：2015-10-01 00:00:00

  • The use of chemical shift temperature gradients to establish the paramagnetic susceptibility tensor orientation: implication for structure determination/refinement in paramagnetic metalloproteins.

    abstract：:The use of dipolar shifts as important constraints in refining molecular structure of paramagnetic metalloproteins by solution NMR is now well established. A crucial initial step in this procedure is the determination of the orientation. of the anisotropic paramagnetic susceptibility tensor in the molecular frame whic...

    journal_title：Journal of biomolecular NMR

    pub_type： 杂志文章

    doi：10.1023/a:1008309121949

    authors： Xia Z,Nguyen BD,La Mar GN

    更新日期：2000-06-01 00:00:00

  • NMR line shape analysis of a multi-state ligand binding mechanism in chitosanase.

    abstract：:Chitosan interaction with chitosanase was examined through analysis of spectral line shapes in the NMR HSQC titration experiments. We established that the substrate, chitosan hexamer, binds to the enzyme through the three-state induced-fit mechanism with fast formation of the encounter complex followed by slow isomeri...

    journal_title：Journal of biomolecular NMR

    pub_type： 杂志文章

    doi：10.1007/s10858-017-0109-6

    authors： Shinya S,Ghinet MG,Brzezinski R,Furuita K,Kojima C,Shah S,Kovrigin EL,Fukamizo T

    更新日期：2017-04-01 00:00:00

  • Application of virus-like particles (VLP) to NMR characterization of viral membrane protein interactions.

    abstract：:The membrane proteins of viruses play critical roles in the virus life cycle and are attractive targets for therapeutic intervention. Virus-like particles (VLP) present the possibility to study the biochemical and biophysical properties of viral membrane proteins in their native environment. Specifically, the VLP cons...

    journal_title：Journal of biomolecular NMR

    pub_type： 杂志文章

    doi：10.1007/s10858-016-0025-1

    authors： Antanasijevic A,Kingsley C,Basu A,Bowlin TL,Rong L,Caffrey M

    更新日期：2016-03-01 00:00:00

  • Numerical design of RN(n)(nu) symmetry-based RF pulse schemes for recoupling and decoupling of nuclear spin interactions at high MAS frequencies.

    abstract：:An approach for the efficient implementation of RN(n)(nu) symmetry-based pulse schemes that are often employed for recoupling and decoupling of nuclear spin interactions in biological solid state NMR investigations is demonstrated at high magic-angle spinning frequencies. RF pulse sequences belonging to the RN(n)(nu) ...

    journal_title：Journal of biomolecular NMR

    pub_type： 杂志文章

    doi：10.1007/s10858-009-9335-x

    authors： Herbst C,Herbst J,Leppert J,Ohlenschläger O,Görlach M,Ramachandran R

    更新日期：2009-08-01 00:00:00

  • Ensembles of a small number of conformations with relative populations.

    abstract：:In our previous work, we proposed a new way to represent protein native states, using ensembles of a small number of conformations with relative Populations, or ESP in short. Using Ubiquitin as an example, we showed that using a small number of conformations could greatly reduce the potential of overfitting and assign...

    journal_title：Journal of biomolecular NMR

    pub_type： 杂志文章

    doi：10.1007/s10858-015-9993-9

    authors： Vammi V,Song G

    更新日期：2015-12-01 00:00:00

  • Optimization of 1H decoupling eliminates sideband artifacts in 3D TROSY-based triple resonance experiments.

    abstract：:TROSY-based triple resonance experiments are essential for protein backbone assignment of large biomolecular systems by solution NMR spectroscopy. In a survey of the current Bruker pulse sequence library for TROSY-based experiments we found that several sequences were plagued by artifacts that affect spectral quality ...

    journal_title：Journal of biomolecular NMR

    pub_type： 杂志文章

    doi：10.1007/s10858-017-0133-6

    authors： Xia Y,Rossi P,Tonelli M,Huang C,Kalodimos CG,Veglia G

    更新日期：2017-09-01 00:00:00

  • Determination of the relative NH proton lifetimes of the peptide analogue viomycin in aqueous solution by NMR-based diffusion measurement.

    abstract：:In aqueous solution, exchanging peptide NH protons experience two environments, that of the peptide itself with a relatively slow diffusion coefficient and that of the water solvent with a faster diffusion coefficient. Although in slow exchange on the NMR chemical shift timescale, the magnetic field gradient dependenc...

    journal_title：Journal of biomolecular NMR

    pub_type： 杂志文章

    doi：10.1023/A:1008393202076

    authors： Liu M,Toms HC,Hawkes GE,Nicholson JK,Lindon JC

    更新日期：1999-01-01 00:00:00

  • Proton, carbon, and nitrogen chemical shifts accurately delineate differences and similarities in secondary structure between the homologous proteins IRAP and IL-1 beta.

    abstract：:1H alpha, 13C alpha, and 15N alpha secondary shifts, defined as the difference between the observed value and the random coil value, have been calculated for interleukin-1 receptor antagonist protein and interleukin-1 beta. Averaging of the secondary chemical shifts with those of adjacent residues was used to smooth o...

    journal_title：Journal of biomolecular NMR

    pub_type： 杂志文章

    doi：10.1007/BF02192848

    authors： Stockman BJ,Scahill TA,Strakalaitis NA,Brunner DP,Yem AW,Deibel MR Jr

    更新日期：1992-11-01 00:00:00

  • Base-type-selective high-resolution 13C edited NOESY for sequential assignment of large RNAs.

    abstract：:Extensive spectral overlap presents a major problem for the NMR study of large RNAs. Here we present NMR techniques for resolution enhancement and spectral simplification of fully 13C labelled RNA. High-resolution 1H-13C correlation spectra are obtained by combining TROSY-type experiments with multiple-band-selective ...

    journal_title：Journal of biomolecular NMR

    pub_type： 杂志文章

    doi：10.1023/a:1008340210079

    authors： Brutscher B,Boisbouvier J,Kupce E,Tisné C,Dardel F,Marion D,Simorre JP

    更新日期：2001-02-01 00:00:00

  • Simultaneous detection of intra- and inter-molecular paramagnetic relaxation enhancements in protein complexes.

    abstract：:Paramagnetic relaxation enhancement (PRE) measurements constitute a powerful approach for detecting both permanent and transient protein-protein interactions. Typical PRE experiments require an intrinsic or engineered paramagnetic site on one of the two interacting partners; while a second, diamagnetic binding partner...

    journal_title：Journal of biomolecular NMR

    pub_type： 杂志文章

    doi：10.1007/s10858-018-0165-6

    authors： Olivieri C,Subrahmanian MV,Xia Y,Kim J,Porcelli F,Veglia G

    更新日期：2018-03-01 00:00:00

  • Automatic NOESY assignment in CS-RASREC-Rosetta.

    abstract：:We have developed an approach for simultaneous structure calculation and automatic Nuclear Overhauser Effect (NOE) assignment to solve nuclear magnetic resonance (NMR) structures from unassigned NOESY data. The approach, autoNOE-Rosetta, integrates Resolution Adapted Structural RECombination (RASREC) Rosetta NMR calcu...

    journal_title：Journal of biomolecular NMR

    pub_type： 杂志文章

    doi：10.1007/s10858-014-9833-3

    authors： Lange OF

    更新日期：2014-07-01 00:00:00

  • Amino-acid type identification in 15N-HSQC spectra by combinatorial selective 15N-labelling.

    abstract：:The efficiency of cell-free protein synthesis combined with combinatorial selective 15N-labelling provides a method for the rapid assignment of 15N-HSQC cross-peaks to the 19 different non-proline amino-acid types from five 15N-HSQC spectra. This strategy was explored with two different constructs of the C-terminal do...

    journal_title：Journal of biomolecular NMR

    pub_type： 杂志文章

    doi：10.1007/s10858-005-5021-9

    authors： Wu PS,Ozawa K,Jergic S,Su XC,Dixon NE,Otting G

    更新日期：2006-01-01 00:00:00

  • NMR View: A computer program for the visualization and analysis of NMR data.

    abstract：:NMR View is a computer program designed for the visualization and analysis of NMR data. It allows the user to interact with a practically unlimited number of 2D, 3D and 4D NMR data files. Any number of spectral windows can be displayed on the screen in any size and location. Automatic peak picking and facilitated peak...

    journal_title：Journal of biomolecular NMR

    pub_type： 杂志文章

    doi：10.1007/BF00404272

    authors： Johnson BA,Blevins RA

    更新日期：1994-09-01 00:00:00

  • Development and application of aromatic [(13)C, (1)H] SOFAST-HMQC NMR experiment for nucleic acids.

    abstract：:Higher sensitivity of NMR spectrometers and novel isotopic labeling schemes have ushered the development of rapid data acquisition methodologies, improving the time resolution with which NMR data can be acquired. For nucleic acids, longitudinal relaxation optimization in conjunction with Ernst angle excitation (SOFAST...

    journal_title：Journal of biomolecular NMR

    pub_type： 杂志文章

    doi：10.1007/s10858-014-9856-9

    authors： Sathyamoorthy B,Lee J,Kimsey I,Ganser LR,Al-Hashimi H

    更新日期：2014-11-01 00:00:00

  • Accessing ns-micros side chain dynamics in ubiquitin with methyl RDCs.

    abstract：:This study presents the first application of the model-free analysis (MFA) (Meiler in J Am Chem Soc 123:6098-6107, 2001; Lakomek in J Biomol NMR 34:101-115, 2006) to methyl group RDCs measured in 13 different alignment media in order to describe their supra-tau (c) dynamics in ubiquitin. Our results indicate that meth...

    journal_title：Journal of biomolecular NMR

    pub_type： 杂志文章

    doi：10.1007/s10858-009-9354-7

    authors： Farès C,Lakomek NA,Walter KF,Frank BT,Meiler J,Becker S,Griesinger C

    更新日期：2009-09-01 00:00:00

  • Improved NMR spectra of a protein-DNA complex through rational mutagenesis and the application of a sensitivity optimized isotope-filtered NOESY experiment.

    abstract：:The NMR spectra of the complex between the DNA-binding domain of the Dead ringer protein (DRI-DBD, Gly262-Gly398) and its DNA binding site (DRI-DBD:DNA, 26 kDa) have been optimized by biochemical and spectroscopic means. First, we demonstrate the utility of a modified 2D [F1,F2] 13C-filtered NOESY experiment that empl...

    journal_title：Journal of biomolecular NMR

    pub_type： 杂志文章

    doi：10.1023/a:1011296112710

    authors： Iwahara J,Wojciak JM,Clubb RT

    更新日期：2001-03-01 00:00:00

  • Application of correlated residual dipolar couplings to the determination of the molecular alignment tensor magnitude of oriented proteins and nucleic acids.

    abstract：:Residual dipolar couplings (RDC) between nuclear spins in partially aligned samples offer unique insights into biomacromolecular structure and dynamics. To fully benefit from the RDC data, accurate knowledge of the magnitude ( D (a)) and rhombicity ( R ) of the molecular alignment tensor, A, is important. An extended ...

    journal_title：Journal of biomolecular NMR

    pub_type： 杂志文章

    doi：10.1023/B:JNMR.0000013701.16162.0c

    authors： Bryce DL,Bax A

    更新日期：2004-03-01 00:00:00

  • An efficient strategy for assignment of cross-peaks in 3D heteronuclear NOESY experiments.

    abstract：:The question is addressed of how maximal structural NOE data on double labelled proteins can be acquired with a minimal set of NOESY experiments. Two 3D-NOESY spectra are reported which, in concert with other commonly used spectra, provide a convenient strategy for NOE assignment. The 3D CNH-NOESY and 3D NCH-NOESY pro...

    journal_title：Journal of biomolecular NMR

    pub_type： 杂志文章

    doi：10.1023/A:1008367912535

    authors： Diercks T,Coles M,Kessler H

    更新日期：1999-10-01 00:00:00

  • Parametrisation of time-averaged distance restraints in MD simulations.

    abstract：:Time-averaged restraints in molecular dynamics simulations offer a means to account for the averaging that is implicit in NMR spectroscopic data. We present a systematic investigation of the parameters which characterise time-averaged distance restraints. Using previously published data for a small protein, chymotryps...

    journal_title：Journal of biomolecular NMR

    pub_type： 杂志文章

    doi：10.1007/BF00197812

    authors： Nanzer AP,van Gunsteren WF,Torda AE

    更新日期：1995-11-01 00:00:00

  • Measuring hydrogen exchange in proteins by selective water saturation in (1)H- (15)N SOFAST/BEST-type experiments: advantages and limitations.

    abstract：:HET(ex)-SOFAST NMR (Schanda et al. in J Biomol NMR 33:199-211, 2006) has been proposed some years ago as a fast and sensitive method for semi-quantitative measurement of site-specific amide-water hydrogen exchange effects along the backbone of proteins. Here we extend this concept to BEST readout sequences that provid...

    journal_title：Journal of biomolecular NMR

    pub_type： 杂志文章

    doi：10.1007/s10858-014-9857-8

    authors： Rennella E,Solyom Z,Brutscher B

    更新日期：2014-11-01 00:00:00