A robust and cost-effective method for the production of Val, Leu, Ile (δ1) methyl-protonated 15N-, 13C-, 2H-labeled proteins (original) (raw)
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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.
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.
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.
Improved strategy for isoleucine 1H/13C methyl labeling in Pichia pastoris
Journal of Biomolecular NMR
Site specific methyl labeling combined with methyl TROSY offers a powerful NMR approach to study structure and dynamics of proteins and protein complexes of high molecular weight. Robust and cost-effective methods have been developed for site specific protein 1 H/ 13 C methyl labeling in an otherwise deuterated background in bacteria. However, bacterial systems are not suitable for expression and isotope labeling of many eukaryotic and membrane proteins. The yeast Pichia pastoris (P. pastoris) is a commonly used host for expression of eukaryotic proteins, and site-specific methyl labeling of perdeuterated eukaryotic proteins has recently been achieved with this system. However, the practical utility of methyl labeling and deuteration in P. pastoris is limited by high costs. Here, we describe an improved method for 1 H/ 13 C-labeling of the δ-methyl group of isoleucine residues in a perdeuterated background, which reduces the cost by ≥ 50% without compromising the efficiency of isotope enrichment. We have successfully implemented this method to label actin and a G-protein coupled receptor. Our approach will facilitate studies of the structure and dynamics of eukaryotic proteins by NMR spectroscopy.
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.
Protein Expression and Purification, 2005
Protocols have been developed and applied for the high-throughput production of [U-15 N]-or [U-13 C-, U-15 N]-labeled proteins using the conditional methionine auxotroph Escherichia coli B834. The large-scale growth and expression uses a chemically defined auto-induction medium containing salts and trace metals, vitamins including vitamin B 12 , and glucose, glycerol, and lactose. The results from nine expression trials in 2-L of the auto-induction medium (500 mL in each of four polyethylene terephthalate beverage bottles) gave an average final optical density at 600 nm of 5,anaveragewetcellmassyieldof5, an average wet cell mass yield of 5,anaveragewetcellmassyieldof9.5 g L À1 , and an average yield of $20 mg of labeled protein in the six instances in which proteolysis of the fusion protein was observed. Correlations between the cell mass recovered, the level of protein expression, and the relative amounts of glucose, glycerol, and lactose in the auto-induction medium were noted. Mass spectral analysis showed that the purified proteins contained both 15 N and 13 C at levels greater than 95%. 1 H-15 N heteronuclear single quantum correlation spectroscopy as well as 13 C; 15 N-edited spectroscopy showed that the purified [U-15 N]-and [U-13 C, U-15 N]-labeled proteins were suitable for structure analysis.
Eur J Biochem, 2001
Bromomethyl ketone derivatives of l-valine (VBMK), l-isoleucine (IBMK), l-norleucine (NleBMK) and l-phenylalanine (FBMK) were synthesized. These reagents were used for qualitative comparative labeling of Escherichia coli valyl-tRNA synthetase (ValRS), an enzyme with Val/Ile editing activity, in order to identify the binding sites for l-valine or noncognate amino acids. Labeling of E. coli ValRS with the substrate analog valyl-bromomethyl ketone (VBMK) resulted in a complete loss of valine-dependent isotopic [ 32 P]PPi±ATP exchange activity. l-Valine protected the enzyme against inactivation. Noncognate amino acids analogs isoleucyl-, norleucyl-and phenylalanyl-bromomethyl ketones (IBMK, NleBMK and FBMK) were also capable of abolishing the activity of ValRS, FBMK being less efficient in inactivating the synthetase. Matrix-assisted laser desorption-ionization mass spectrometry designated cysteines 424 and 829 as the target residues of the substrate analog VBMK on E. coli ValRS, whereas, altogether, IBMK, NleBMK and FBMK labeled His266, Cys275, His282, His433 and Cys829, of which Cys275, His282 and His433 were labeled in common by all three noncognate amino-acid-derived bromomethyl ketones. With the exception of Cys829, which was most likely unspecifically labeled, the amino-acid residues labeled by the reagents derived from noncognate amino acids were distributed between two fragments 259±291 and 419±434 in the primary structure of E. coli ValRS. In fragment 419±434, Cys424 was specifically labeled by the substrate analog VBMK, while His433 was labeled in common by all the used bromomethyl ketone derivatives of noncognate amino acids, suggesting that the synthetic site where aminoacyl adenylate formation takes place on E. coli ValRS is built up of two subsites. One subsite containing Cys424 might represent the catalytic locus of the active center where specific l-valine activation takes place. The second subsite containing His433 might represent the binding site for noncognate amino acids. The fact that Cys275 and His282, fragment 259±291, were labeled by IBMK, NleBMK and FBMK, but not by the substrate analog VBMK, suggests that these residues might be located at or near the editing site of E. coli ValRS. Comparison of fragment 259±291 with all the available ValRS amino-acid sequences revealed that His282 is strictly conserved, with the exception of its replacement by a glycine in a subgroup corresponding to the archaebacteria. Because a nucleophile is needed in the editing site to achieve hydrolysis of an undesired product at the level of the carbonyl group thereof, it is proposed that the conserved His282 of E. coli ValRS is involved in editing.
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
Protein Expression and Purification, 2009
Pseudomonas fluorescens is a robust protein expression system that is very well suited for high throughput protein expression for structural genomics studies. Since NMR spectroscopy and X-ray crystallography are both used by various investigators in structure elucidation studies, the availability of target proteins labeled with stable isotopes or selenomethionine is essential for the determination of protein structures. A completely defined medium for the expression and stable isotope labeling of proteins in P. fluorescens has been developed. The expression level of Bacillus thuringiensis Cry34 in the modified medium is comparable to that obtained in the original medium. In addition, more than 95% incorporation of 15 N was obtained in Cry34 using 15 N ammonium sulfate and the quality of the protein, as assessed by NMR analysis, is comparable to that made using commercial medium. High levels of selenomethionine (SeMet) incorporation in the Xenorhabdus nematophilus insecticidal protein XptA2 were also obtained in P. fluorescens using the defined medium, allowing development of a method for obtaining highly purified XptA2. The following observations were made when inhibitors of endogenous methionine biosynthesis were used in P. fluorescens culture when SeMet was substituted in XptA2: (I) there is little inhibition of cell growth or recombinant XptA2 expression in the presence of SeMet concentrations up to 300 mg/L in cell culture, (II) there was greater than 95% SeMet incorporation ratio in recombinant SeMet-labeled XptA2 (SeMet-XptA2) and the incorporation ratio is consistent and reproducible and (III) finally, purified SeMet-XptA2 possesses similar protein structure and insecticidal activity relative to the unlabeled counterpart XptA2 as shown by bioassay and differential scanning calorimetric analysis. The high SeMet incorporation should provide high accuracy and resolution in XptA2 phase determination by multiwavelength anomalous diffraction (MAD), indicating that P. fluorescens is an excellent expression host to produce SeMet-labeled proteins for structural study.