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Volumn 39, Issue 12, 2000, Pages 2148-2152

Expanding the scope of protein biosynthesis by altering the methionyl- tRNA synthetase activity of a bacterial expression host

Author keywords

Amino acids; Biosynthesis; Ligases; Protein expression; Protein modifications

Indexed keywords

METHIONINE TRANSFER RNA LIGASE;

EID: 0034674281     PISSN: 14337851     EISSN: None     Source Type: Journal    
DOI: 10.1002/1521-3773(20000616)39:12<2148::AID-ANIE2148>3.0.CO;2-7     Document Type: Article
Times cited : (101)

References (60)
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    • Chemical aminoacylation methods circumvent the AARS altogether (S. M. Hecht, B. L. Alford, Y. Kuroda, S. Kitano, J. Biol. Chem. 1978, 253, 4517-4520; C. J. Noren, S. J. Anthony-Cahill, M. C. Griffith, P. G. Schultz, Science 1989, 244, 182-188; J. D. Bain, C. G. Glabe, T. A. Dix, A. R. Chamberlin, J. Am. Chem. Soc. 1989, 111, 8013-8014) but suffer from low protein yields. Alteration of the AARS activities of the cell through introduction of heterologous synthetases (D. R. Liu, P. G. Schultz, Proc. Natl. Acad. Sci. USA 1999, 96, 4780-4785; R. Furter, Protein Sci. 1998, 7, 419-426; D. R. Liu, T. J. Magliery, M. Pastrnak, P. G. Schultz, Proc. Natl. Acad. Sci. USA 1997, 94, 10092-10097) is also possible. The relative simplicity of the in vivo approach, its capacity for multisite substitution, and its high synthetic efficiency make it the method of choice whenever possible.
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    • Chemical aminoacylation methods circumvent the AARS altogether (S. M. Hecht, B. L. Alford, Y. Kuroda, S. Kitano, J. Biol. Chem. 1978, 253, 4517-4520; C. J. Noren, S. J. Anthony-Cahill, M. C. Griffith, P. G. Schultz, Science 1989, 244, 182-188; J. D. Bain, C. G. Glabe, T. A. Dix, A. R. Chamberlin, J. Am. Chem. Soc. 1989, 111, 8013-8014) but suffer from low protein yields. Alteration of the AARS activities of the cell through introduction of heterologous synthetases (D. R. Liu, P. G. Schultz, Proc. Natl. Acad. Sci. USA 1999, 96, 4780-4785; R. Furter, Protein Sci. 1998, 7, 419-426; D. R. Liu, T. J. Magliery, M. Pastrnak, P. G. Schultz, Proc. Natl. Acad. Sci. USA 1997, 94, 10092-10097) is also possible. The relative simplicity of the in vivo approach, its capacity for multisite substitution, and its high synthetic efficiency make it the method of choice whenever possible.
    • (1989) Science , vol.244 , pp. 182-188
    • Noren, C.J.1    Anthony-Cahill, S.J.2    Griffith, M.C.3    Schultz, P.G.4
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    • Chemical aminoacylation methods circumvent the AARS altogether (S. M. Hecht, B. L. Alford, Y. Kuroda, S. Kitano, J. Biol. Chem. 1978, 253, 4517-4520; C. J. Noren, S. J. Anthony-Cahill, M. C. Griffith, P. G. Schultz, Science 1989, 244, 182-188; J. D. Bain, C. G. Glabe, T. A. Dix, A. R. Chamberlin, J. Am. Chem. Soc. 1989, 111, 8013-8014) but suffer from low protein yields. Alteration of the AARS activities of the cell through introduction of heterologous synthetases (D. R. Liu, P. G. Schultz, Proc. Natl. Acad. Sci. USA 1999, 96, 4780-4785; R. Furter, Protein Sci. 1998, 7, 419-426; D. R. Liu, T. J. Magliery, M. Pastrnak, P. G. Schultz, Proc. Natl. Acad. Sci. USA 1997, 94, 10092-10097) is also possible. The relative simplicity of the in vivo approach, its capacity for multisite substitution, and its high synthetic efficiency make it the method of choice whenever possible.
    • (1989) J. Am. Chem. Soc. , vol.111 , pp. 8013-8014
    • Bain, J.D.1    Glabe, C.G.2    Dix, T.A.3    Chamberlin, A.R.4
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    • 0033609150 scopus 로고    scopus 로고
    • Chemical aminoacylation methods circumvent the AARS altogether (S. M. Hecht, B. L. Alford, Y. Kuroda, S. Kitano, J. Biol. Chem. 1978, 253, 4517-4520; C. J. Noren, S. J. Anthony-Cahill, M. C. Griffith, P. G. Schultz, Science 1989, 244, 182-188; J. D. Bain, C. G. Glabe, T. A. Dix, A. R. Chamberlin, J. Am. Chem. Soc. 1989, 111, 8013-8014) but suffer from low protein yields. Alteration of the AARS activities of the cell through introduction of heterologous synthetases (D. R. Liu, P. G. Schultz, Proc. Natl. Acad. Sci. USA 1999, 96, 4780-4785; R. Furter, Protein Sci. 1998, 7, 419-426; D. R. Liu, T. J. Magliery, M. Pastrnak, P. G. Schultz, Proc. Natl. Acad. Sci. USA 1997, 94, 10092-10097) is also possible. The relative simplicity of the in vivo approach, its capacity for multisite substitution, and its high synthetic efficiency make it the method of choice whenever possible.
    • (1999) Proc. Natl. Acad. Sci. USA , vol.96 , pp. 4780-4785
    • Liu, D.R.1    Schultz, P.G.2
  • 22
    • 0031937870 scopus 로고    scopus 로고
    • Chemical aminoacylation methods circumvent the AARS altogether (S. M. Hecht, B. L. Alford, Y. Kuroda, S. Kitano, J. Biol. Chem. 1978, 253, 4517-4520; C. J. Noren, S. J. Anthony-Cahill, M. C. Griffith, P. G. Schultz, Science 1989, 244, 182-188; J. D. Bain, C. G. Glabe, T. A. Dix, A. R. Chamberlin, J. Am. Chem. Soc. 1989, 111, 8013-8014) but suffer from low protein yields. Alteration of the AARS activities of the cell through introduction of heterologous synthetases (D. R. Liu, P. G. Schultz, Proc. Natl. Acad. Sci. USA 1999, 96, 4780-4785; R. Furter, Protein Sci. 1998, 7, 419-426; D. R. Liu, T. J. Magliery, M. Pastrnak, P. G. Schultz, Proc. Natl. Acad. Sci. USA 1997, 94, 10092-10097) is also possible. The relative simplicity of the in vivo approach, its capacity for multisite substitution, and its high synthetic efficiency make it the method of choice whenever possible.
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    • 0030984176 scopus 로고    scopus 로고
    • Chemical aminoacylation methods circumvent the AARS altogether (S. M. Hecht, B. L. Alford, Y. Kuroda, S. Kitano, J. Biol. Chem. 1978, 253, 4517-4520; C. J. Noren, S. J. Anthony-Cahill, M. C. Griffith, P. G. Schultz, Science 1989, 244, 182-188; J. D. Bain, C. G. Glabe, T. A. Dix, A. R. Chamberlin, J. Am. Chem. Soc. 1989, 111, 8013-8014) but suffer from low protein yields. Alteration of the AARS activities of the cell through introduction of heterologous synthetases (D. R. Liu, P. G. Schultz, Proc. Natl. Acad. Sci. USA 1999, 96, 4780-4785; R. Furter, Protein Sci. 1998, 7, 419-426; D. R. Liu, T. J. Magliery, M. Pastrnak, P. G. Schultz, Proc. Natl. Acad. Sci. USA 1997, 94, 10092-10097) is also possible. The relative simplicity of the in vivo approach, its capacity for multisite substitution, and its high synthetic efficiency make it the method of choice whenever possible.
    • (1997) Proc. Natl. Acad. Sci. USA , vol.94 , pp. 10092-10097
    • Liu, D.R.1    Magliery, T.J.2    Pastrnak, M.3    Schultz, P.G.4
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    • 1) (H. Y. Kim, G. Ghosh, L. H. Schulman, S. Brunie, H. Jakubowski, Proc Natl. Acad. Sci. USA 1993, 90, 11553-11557). Kinetic constants were calculated by nonlinear regression analysis.
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    • A gene encoding a mutant MetRS was removed from plasmid pBSM547W305F (D. Fourmy, Y. Mechulam, S. Brunie, S. Blanquet, G. Fayat, FEBS Lett. 1991, 292, 259-263) by treatment with restriction enzymes Sac I and Kpn I. The Sac I/Kpn I fragment (2450 bp) was ligated into the cloning vector pUC19-Nhelink, which was constructed to permit the cohesive ends of the mutant MetRS gene to be changed to Nhe I. The MetRS gene with Nhe I cohesive ends was then ligated into the unique Nhe I site of the plasmid pQE15 (Qiagen, Inc., Valencia, CA, USA) to yield plasmid pQEI5-W305F. Transformation of pQE15-W305F into a rec A-positive cell strain resulted in genetic recombination of the mutant MetRS gene with the chromosomal copy of the wild-type MetRS gene, yielding plasmid pQE15-MRS. Expression plasmid pQE15-MRS and repressor plasmid pREP4 were transformed into the expression host B834(DE3) to yield B834(DE3)/ pQE15-MRS/pREP4. Plasmid DNA from all B834(DE3)/pQE15-MRS/pREP4 cultures used for protein expression experiments was sequenced to confirm that it encoded wild-type MetRS.
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    • Fourmy, D.1    Mechulam, Y.2    Brunie, S.3    Blanquet, S.4    Fayat, G.5
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    • The Qiagen Expressionist, Qiagen, Inc., Valencia, CA, 2000, p. 68.
    • (2000) The Qiagen Expressionist , pp. 68
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    • [13]
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    • 1, respectively.
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    • B834(DE3)/pQE15-MRS/pREP4 cells, which overexpress MetRS, have sufficient MetRS activity to synthesize measurable levels of protein from the very low intracellular levels of methionine in the negative-control culture. Interestingly, AARS overexpression is induced by amino acid starvation in some Gram-positive bacteria, presumably to permit continued protein synthesis (D. Luo, J. Leautey, M. Grunberg-Manago, H. Putzer, Bacterial. 1997, 179, 2472-2478). B834(DE3)/pOE15/pREP4 cultures, which lack the increased MetRS activity, do not show background expression of protein in negative-control cultures.
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    • Luo, D.1    Leautey, J.2    Grunberg-Manago, M.3    Putzer, H.4
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    • 0342477956 scopus 로고    scopus 로고
    • note
    • 60D of 20 was prepared by one freeze - thaw cycle and added to the assay mixture to yield a final volume of 150 μL. A saturating concentration of methionine (750 μM) was used to determine the maximum exchange velocity for each cell lysate.
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    • 0343783114 scopus 로고    scopus 로고
    • note
    • 1H NMR spectra were recorded by using a Varian Inova NMR spectrometer with proton acquisition at 599.69 MHz. Spectra were recorded at 25 °C overnight. A simple presaturation pulse was used for water suppression.
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    • 0003180169 scopus 로고    scopus 로고
    • 1D TOCSY NMR spectra were recorded on a Varian Inova NMR spectrometer with proton acquisition at 599.69 MHz. A 1D TOCSY pulse sequence (D. Uhrin, P. N. Barlow, J. Magn. Reson. 1997, 126, 248-255) with selective irradiation of the signal at δ = 5.35 (E. Kupce, J. Boyd, I. D. Campbell, J. Magn. Reson. Ser. B. 1995, 106, 300-303) was used to identify which protons belonged to the spin system of that signal. The selectivity of the pulse is demonstrated in a separate, simple 1D experiment in which the selective pulse is applied alone; no other resonances are observed in the spectrum under these conditions. Observation after a mixing time of 60 ms, however, showed the protons at δ = 5.60 and 5.70, indicating that those protons are members of the same spin system (and therefore the same amino acid residue) as those corresponding to the resonance at δ = 5.35. The α-carbon and side chain β- and ε-carbon protons are also observed at chemical shift values characteristic of the free amino acid (δ = 4.3 (α-CH), 2.5 (β-CH2), and 1.6 (δ-CH3)).
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    • Uhrin, D.1    Barlow, P.N.2
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    • 0029270246 scopus 로고
    • 1D TOCSY NMR spectra were recorded on a Varian Inova NMR spectrometer with proton acquisition at 599.69 MHz. A 1D TOCSY pulse sequence (D. Uhrin, P. N. Barlow, J. Magn. Reson. 1997, 126, 248-255) with selective irradiation of the signal at δ = 5.35 (E. Kupce, J. Boyd, I. D. Campbell, J. Magn. Reson. Ser. B. 1995, 106, 300-303) was used to identify which protons belonged to the spin system of that signal. The selectivity of the pulse is demonstrated in a separate, simple 1D experiment in which the selective pulse is applied alone; no other resonances are observed in the spectrum under these conditions. Observation after a mixing time of 60 ms, however, showed the protons at δ = 5.60 and 5.70, indicating that those protons are members of the same spin system (and therefore the same amino acid residue) as those corresponding to the resonance at δ = 5.35. The α-carbon and side chain β- and ε-carbon protons are also observed at chemical shift values characteristic of the free amino acid (δ = 4.3 (α-CH), 2.5 (β-CH2), and 1.6 (δ-CH3)).
    • (1995) J. Magn. Reson. Ser. B. , vol.106 , pp. 300-303
    • Kupce, E.1    Boyd, J.2    Campbell, I.D.3
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    • note
    • The signal corresponding to methionine elutes at 13.8 min while that corresponding to Tcg elutes at 16.0 min. The large peaks which elute at approximately 15.4 min correspond to piperidylphenylthiourea (pptu), a product of the analysis resulting from the buffer, and the small peak at 19.4 min corresponds to diethylphthalate (diet), an internal standard.
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    • H. E. Blackwell, R. H. Grubbs, Angew. Chem. 1998, 110, 3469-3472; Angew. Chem. Int. Ed. 1998, 37, 3281-3284.
    • (1998) Angew. Chem. Int. Ed. , vol.37 , pp. 3281-3284


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