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This paper describes the crystal structure of modified glutathione transferase where all the tyrosine residues are replaced by its analog 3-fluorotyrosine. This is one of the first reports of analysis of a protein structure with such a fluorotyrosine substitution.
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This paper describes a procedure for preparing recombinant protein by in vivo synthesis using an auxotropic strain of E. coli and special growth media, where all the proline residues in the protein are replaced by the proline analog thiaproline. Such proteins are very useful for biophysical analysis.
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23
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This paper describes two methods for stable isotope labeling of proteins using cell-free translation. One special feature of this work is the utilization of a high yield optimized cell-free translation system. For example, 2.2 mg of site-directed isotope labeled (SDIL) c-Ha-Ras protein was synthesized from a 30 ml reaction system for the purpose of NMR analysis.
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Yabuki T, Kigawa T, Dohmae N, Takio K, Terada T, Ito Y, Laue ED, Cooper JA, Kainosho M, Yokoyama S Dual amino acid-selective and site-directed stable-isotope labeling of the human c-Ha-Ras protein by cell-free synthesis. J Biomol NMR. 11:1998;295-306. This paper describes two methods for stable isotope labeling of proteins using cell-free translation. One special feature of this work is the utilization of a high yield optimized cell-free translation system. For example, 2.2 mg of site-directed isotope labeled (SDIL) c-Ha-Ras protein was synthesized from a 30 ml reaction system for the purpose of NMR analysis.
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24
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This paper describes a novel method of in vitro translation that permits the isotope labeling of specific regions of a protein (region-directed isotope labeling). The translation is carried out using column bound mRNA and a specialized translation mix which is designed for labeling the particular region of the protein.
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Pavlov MY, Freistroffer DV, Ehrenberg M Synthesis of region labeled proteins for MNR studies by in vitro translation of column-coupled mRNAs. Biochimie. 79:1997;415-422. This paper describes a novel method of in vitro translation that permits the isotope labeling of specific regions of a protein (region-directed isotope labeling). The translation is carried out using column bound mRNA and a specialized translation mix which is designed for labeling the particular region of the protein.
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Nowak MW, Gallivan JP, Silverman SK, Labarca CG, Dougherty DA, Lester HA In vivo incorporation of unnatural amino acids into ion channels in Xenopus oocyte expression system. Methods Enzymol. 293:1998;504-529. This report describes in vivo SNAAR using the Xenopus oocyte system. The authors also address the feasibility of incorporating non-native amino acids into ion channels and membrane receptors in mammalian cells.
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26
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Liu DR, Magliery TJ, Pastrnak M, Schultz PG Engineering a tRNA and aminoacyl-tRNA synthetase for the site-specific incorporation of unnatural amino acids into proteins in vivo. Proc Natl Acad Sci USA. 94:1997;10092-10097. This article describes the construction of a novel tRNA-aminoacyl-tRNA synthetase pair by genetic manipulation and its use for in vivo site-specific incorporation of non-native amino acids into proteins.
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31
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This paper describes the incorporation of fluorescent amino acid analogs into β-galactosidase using a translation system derived from a special E. coli strain that is defective in one of the release factors. The authors describe how such a system may improve suppression efficiency for SNAAR.
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Steward LE, Collins CS, Gilmore MA, Carlson JE, Ross JBA, Chamberlin AR In vitro site-specific incorporation of fluorescent probes into β-galactosidase. J Am Chem Soc. 119:1997;6-11. This paper describes the incorporation of fluorescent amino acid analogs into β-galactosidase using a translation system derived from a special E. coli strain that is defective in one of the release factors. The authors describe how such a system may improve suppression efficiency for SNAAR.
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Gallivan JP, Lester HA, Dougherty DA Site-specific incorporation of biotinylated amino acids to identify surface-exposed residues in integral membrane proteins. Chem Biol. 4:1997;739-749. The authors describe the use of biotin residues incorporated by SNAAR into the nicotinic acetylcholine receptor for probing surface-exposed regions. This approach should be very useful to explore the topology of a wide range of receptors and serve as an alternative to more conventional strategies, such as the cysteine substitution approach.
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Blanquet, S.3
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US Patent 1997, no 5 654 722. Methods are disclosed for the non-radioactive labeling, detection, quantitation and isolation of nascent proteins translated in a cellular or cell-free translation system. Use of misaminoacylated tRNA molecules facilitates the detection and isolation of nascent protein from other components of the translation system
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Rothschild KJ, Olejnik J, Sonar S: Methods for the detection and isolation of proteins. US Patent 1997, no 5 654 722. Methods are disclosed for the non-radioactive labeling, detection, quantitation and isolation of nascent proteins translated in a cellular or cell-free translation system. Use of misaminoacylated tRNA molecules facilitates the detection and isolation of nascent protein from other components of the translation system.
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Methods for the Detection and Isolation of Proteins
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Rothschild, K.J.1
Olejnik, J.2
Sonar, S.3
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