Summary
A cell-free translation system producing mature green fluorescent protein (GFP) can be a useful tool for studying the mechanism and kinetics of GFP chromophore formation, as well as for fast protein engineering. We report here that the mature GFP can be formed in the cell-free translation system from E.coli. The synthesis of GFP in the cell-free system reaches a plateau in 30 to 40 min whereas its maturation is completed in 4 h from the beginning of translation. The delay between the GFP synthesis and the chromophore formation in the cell-free system provides the possibility to isolate and to analyse maturation intermediates for elucidation of the modification pathway.
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Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W. and Prasher, D. C. (1994). Science 263, 802–805.
Chen, H.-Z. and Zubay, G. (1983). Methods Enzymol. 101, 674–690.
Cody, C. W., Prasher, D. C., Westler, W. M., Prendergast, F. G. and Ward, W. W. (1993). Biochemistry 32, 1212–1218.
Cubitt, A. B., Heim, R., Adams S. R., Boyd, A. E., Gross, L. A., and Tsien, R. Y. (1995). Trends Biochem. Sci. 20, 448–455.
Gurevich, V.V., Pokrovskaya, I.D., Obukhova, T.A. and Zozulya, S.A. (1991). Anal. Biochem. 195, 207–213.
Heim, R., Cubitt, A. B. and Tsien, R. Y. (1995). Nature (London) 373, 663–664.
Heim, R., Prasher, D. C. and Tsien, R. (1994). Proc.Natl.Acad.Sci. USA. 91, 12501–12504.
Inouye, S. and Tsuji, F.I. (1994). FEBS Lett. 341, 277–280.
Laemmli, U. K. (1970). Nature (London) 227, 680–685.
Morise, H., Shimomura, O., Johnson, F. H. and Winant, J. (1974). Biochemistry 13, 2656–2662.
Prasher, D. C., Eckenrode, V. K., Ward, W. W., Prendergast, F. G. and Cormier, M. G. (1992). Gene 111, 229–233.
Shimomura, O. (1979). FEBS Lett. 104, 220–222.
Wang, S. and Hazelrigg, T. (1994) Nature (London) 369, 400–403.
Ward, W. W. (1979). In: Photochemical and Photobiological Reviews, Smith, K. C. ed., Vol. 4, 1–57, New York: Plenum.
Ward, W. W. and Bokman, S. H. (1982). Biochemistry 21, 4535–4540.
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Kolb, V.A., Makeyev, E.V., Ward, W.W. et al. Synthesis and maturation of green fluorescent protein in a cell-free translation system. Biotechnol Lett 18, 1447–1452 (1996). https://doi.org/10.1007/BF00129353
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DOI: https://doi.org/10.1007/BF00129353