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Kariya, Eri; Ohki, Shinya; Hayano, Toshiya; Kainosho, Masatsune

doi:10.1023/a:1008314730151

Cited by 15 publications

(19 citation statements)

References 4 publications

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“…1 H resonances in PpiB [23], and the availability of those data permitted the immediate assignment of most of the cysteine and phenylalanine amide resonances in the spectra of Fig. 4A.…”

Section: N-mentioning

confidence: 98%

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Translational incorporation of L‐3,4‐dihydroxyphenylalanine into proteins

et al. 2005

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The incorporation of non-natural amino acids opens up the possibility to endow proteins with properties that cannot be attained with the 20 natural amino acids encoded by DNA base triplets. The incorporation of non-natural amino acids can readily be achieved with the natural protein-translational machinery, if the structure of the modified amino acid is closely related to the natural amino acid, so that it can be loaded onto tRNA by one of the natural aminoacyl-tRNA synthetases.A wide range of non-natural amino acids has been incorporated into proteins in this way [1]. In general, the efficiency of incorporation decreases with increasing K M value of the aminoacyl-tRNA synthetase for the respective amino acid. This holds, in particular, for the in vivo incorporation of non-natural amino acids, where a pool of natural amino acids is always present. This problem can be circumvented by the use of auxotrophic strains [1] or cell-free protein production systems derived from nonauxotrophic strains combined with a suitably manipulated medium for protein synthesis [2,3].Recently, high-yield, cell-free protein production systems have become available that allow the synthesis of proteins in quantities sufficient for structural genomics applications [4][5][6][7]. High-level incorporation of seleno-methionine (Se-Met) for X-ray crystallography and fluoro-tryptophan (F-Trp) for NMR has been An Escherichia coli cell-free transcription ⁄ translation system was used to explore the high-level incorporation of l-3,4-dihydroxyphenylalanine (DOPA) into proteins by replacing tyrosine with DOPA in the reaction mixtures. ESI-MS showed specific incorporation of DOPA in place of tyrosine. More than 90% DOPA incorporation at each tyrosine site was achieved, allowing the recording of clean 15 N-HSQC NMR spectra. A redox-staining method specific for DOPA was shown to provide a sensitive and generally applicable method for assessing the cell-free production of proteins. Of four proteins produced in soluble form in the presence of tyrosine, two resulted in insoluble aggregates in the presence of high levels of DOPA. DOPA has been found in human proteins, often in association with various disease states that implicate protein aggregation and ⁄ or misfolding. Our results suggest that misfolded and aggregated proteins may result, in principle, from ribosome-mediated misincorporation of intracellular DOPA accumulated due to oxidative stress. High-yield cell-free protein expression systems are uniquely suited to obtain rapid information on solubility and aggregation of nascent polypeptide chains.Abbreviations DOPA, L-3,4-dihydroxyphenylalanine; GFP, cycle 3 mutant green fluorescent protein; hCypA, human cyclophilin A; His 6 -PpiB, N-terminal His 6 -tagged PpiB; HMP, Escherichia coli flavohaemoglobin; HSQC, heteronuclear single-quantum coherence; NBT, nitroblue tetrazolium; PpiB, E. coli peptidyl-prolyl cis-trans isomerase B; RNAP, RNA polymerase; TyrRS, tyrosyl-tRNA synthetase.

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“…1 H resonances in PpiB [23], and the availability of those data permitted the immediate assignment of most of the cysteine and phenylalanine amide resonances in the spectra of Fig. 4A.…”

Section: N-mentioning

confidence: 98%

“…1EMA) [22]. The first two proteins had previously been shown to be produced in good yield in the in vitro reaction [5,6,18,23].…”

Section: Protein Synthesis In the Presence Of Dopamentioning

confidence: 99%

Translational incorporation of L‐3,4‐dihydroxyphenylalanine into proteins

et al. 2005

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“…A meaningful NMR analysis is possible straight from the reaction mixture and spectra containing only the signals from the in vitro synthesized protein can be obtained after simple dialysis to remove lowmolecular weight metabolites (17). In a related fashion, fluorotryptophan has been incorporated into proteins for subsequent analysis by 19 F-NMR spectroscopy (31). This simplicity invites applications in high-throughput mode.…”

Section: Simplicitymentioning

confidence: 99%

“…Complete elimination of these metabolic enzymes to achieve high efficiency and selectivity of isotope labeling can be achieved by complete reconstitution of the cell-free system with purified components, as in the 'PURE' system (30), but this appears uneconomical and unnecessary for the purpose of 15 N-labeling; the transaminase activities in conventional S30 extracts from non-auxotrophic E. coli strains can be suppressed sufficiently to yield clean 15 N-HSQC spectra without any sign of cross-labeling (17,33). Transaminase activities are of course of no concern when uniformly isotope-labeled proteins are produced (18,19,32,35).…”

Section: Efficiency and Importance Of S30 Extractsmentioning

confidence: 99%

“…Complete incorporation of fluoro-tryptophan in place of tryptophan has been demonstrated (31). Since fluorine is not otherwise present in the reaction medium, 19 F-NMR offers a sensitive way of determining whether a protein is folded or unfolded without prior purification of the protein (31). In other cases, the isotopelabeled amino acids added to the cell-free reaction medium were found to be diluted by a few percent of unlabeled amino acids that came with the S30 extract (35).…”

Section: Incorporation Of Non-natural Amino Acidsmentioning

confidence: 99%

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Cell-free synthesis of 15 N-labeled proteins for NMR studies

Ozawa

Dixon

Otting

2005

IUBMB Life (International Union of Biochemistry and Molecular B

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SummaryModern cell-free in vitro protein synthesis systems present powerful tools for the synthesis of isotope-labeled proteins in high yields. The production of selectively 15 N-labeled proteins from 15 Nlabeled amino acids is particularly economic and yields are often sufficient to analyze the proteins very quickly by two-dimensional NMR spectra recorded of the crude reaction mixture without concentration or chromatographic purification of the protein. We review methodological aspects of cell-free in vitro protein synthesis based on an Escherichia coli cell extract, in particular with regard to the production of 15 N-labeled proteins for analysis by NMR spectroscopy.IUBMB Life, 57: 615 -622, 2005 Keywords Cell-free protein synthesis; E. coli S30 extract; 15 Nlabeling; NMR spectroscopy; 15 N-HSQC spectrum.

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Efficient production of isotopically labeled proteins by cell-free synthesis: A practical protocol

et al. 2004

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We provide detailed descriptions of our refined protocols for the cell-free production of labeled protein samples for NMR spectroscopy. These methods are efficient and overcome two critical problems associated with the use of conventional Escherichia coli extract systems. Endogenous amino acids normally present in E. coli S30 extracts dilute the added labeled amino acids and degrade the quality of NMR spectra of the target protein. This problem was solved by altering the protocol used in preparing the S30 extract so as to minimize the content of endogenous amino acids. The second problem encountered in conventional E. coli cell-free protein production is non-uniformity in the N-terminus of the target protein, which can complicate the NMR spectra. This problem was solved by adding a DNA sequence to the construct that codes for a cleavable N-terminal peptide tag. Addition of the tag serves to increase the yield of the protein as well as to ensure a homogeneous protein product following tag cleavage. We illustrate the method by describing its stepwise application to the production of calmodulin samples with different stable isotope labeling patterns for NMR analysis.

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Cited by 15 publications

References 4 publications

Translational incorporation of L‐3,4‐dihydroxyphenylalanine into proteins

Translational incorporation of L‐3,4‐dihydroxyphenylalanine into proteins

Cell-free synthesis of 15 N-labeled proteins for NMR studies

Efficient production of isotopically labeled proteins by cell-free synthesis: A practical protocol

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