Abstract
A new strategy is described for the production of peptides enriched with stable isotopes. Peptides of interest are expressed in Escherichia coli (E. coli) cells as recombinant fusion proteins with Saccharomyces cerevisiae ubiquitin. This method yields as much as 30–100 mg/l of isotope-enriched fusion proteins in minimal media. A decahistidine tag attached to the N-terminus of ubiquitin enables a one-step purification of the fusion protein via Ni2+-chelating affinity chromatography. The ubiquitin moiety is then easily and specifically cleaved off by a protease, yeast ubiquitin hydrolase. Since this enzyme is also expressed at a high level in E. coli cells and can be purified in one step, the presented strategy has an advantage in view of costs over others that use commercially available proteases. In addition, since ubiquitin fusion proteins easily refold, the fusion protein can be expressed either in a soluble form or as inclusion bodies. This flexibility enables us to prepare peptides that are unstable in a soluble state in E. coli cells. As an example, the expression and the uniform stable isotope enrichment with 15N and/or13 C are described for mastoparan-X, a tetradecapeptide known to activate GTP-binding regulatory proteins. An amide group at the C-terminus of this peptide can also be formed by our method. The presented system is considered powerful for the stable isotope enrichment of short peptides with proton resonances that are too severely overlapped to be analyzed solely by proton NMR.
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Andrieux, M., Leroy, E., Guittet, E., Ritco-Vonsovici, M., Mouratou, B., Minard, P., Desmadril, M. and Yon, J.M. (1995) Biochemistry, 34, 842–846.
Anglister, J., Scherf, T., Zilber, B. and Levy, R. (1995) Biopolymers, 37, 383–389.
Bax, A. and Davis, D.G. (1985) J. Magn. Reson., 65, 355–359.
Bax, A. and Grzesiek, S. (1993) Acc. Chem. Res., 26, 131–138.
Bodenhausen, G. and Ruben, D.J. (1980) Chem. Phys. Lett., 69, 185–189.
Campbell, A.P. and Sykes, B.D. (1991) J. Magn. Reson., 93, 77–92.
Campbell, A.P. and Sykes, B.D. (1993) Annu. Rev. Biophys. Biomol. Struct., 22, 99–122.
Campbell, A.P., Wong, W.Y., Houston, Jr., M., Schweizer, F., Cachia, P.J., Irvin, R.T., Hindsgaul, O., Hodges, R.S. and Sykes, B.D. (1997) J. Mol. Biol., 267, 382–402.
Carpenter, K.A. and Ni, F. (1992) J. Magn. Reson., 99, 192–197.
Clore, G.M. and Gronenborn, A.M. (1982) J. Magn. Reson., 48, 402–417.
Clore, G.M. and Gronenborn, A.M. (1983) J. Magn. Reson., 53, 423–442.
Clore, G.M. and Gronenborn, A.M. (1991) Prog. NMR Spectrosc., 23, 43–92.
Cordier-Ochsenbein, F., Guerois, R., Baleux, F., Huynh-Dinh, T., Chaffotte, A., Neumann, J.M. and Sanson, A. (1996) Biochemistry, 35, 10347–10357.
Delaglio, F., Grzesiek, S., Vuister, G.W., Zhu, G., Pfeifer, J. and Bax, A. (1995) J. Biomol. NMR, 6, 277–293.
Gilman, A.G. (1987) Annu. Rev. Biochem., 56, 615–649.
Grzesiek, S. and Bax, A. (1992) J. Am. Chem. Soc., 114, 6291–6293.
Grzesiek, S. and Bax, A. (1993) J. Am. Chem. Soc., 115, 12593–12594.
Henry, G.D. and Sykes, B.D. (1992) Biochemistry, 31, 5284–5297.
Higashijima, T., Uzu, S., Nakajima, T. and Ross, E.M. (1988) J. Biol. Chem., 263, 6491–6494.
Ikura, M., Bax, A., Clore, G.M. and Gronenborn, A.M. (1990) J. Am. Chem. Soc., 112, 9020–9022.
Jahnke, W., Baur, M., Gemmecker, G. and Kessler, H. (1995) J. Magn. Reson., B106, 86–88.
Jelinek, R., Terry, T.D., Gesell, J.J., Malik, P., Perham, R.N. and Opella, S.J. (1997) J. Mol. Biol., 266, 649–655.
Katopodis, A.G., Ping, D. and May, S.W. (1990) Biochemistry, 29, 6115–6120.
Kay, L.E., Ikura, M., Tschudin, R. and Bax, A. (1990) J. Magn. Reson., 89, 496–514.
Kusunoki, H., Wakamatsu, K., Sato, K., Miyazawa, T. and Kohno, T. (1998) Biochemistry, 37, 4782–4791.
MacKenzie, K.R., Prestegard, J.H. and Engelman, D.M. (1997) Science, 276, 131–133.
Marion, D. and Wüthrich, K. (1983) Biochem. Biophys. Res. Commun., 113, 967–974.
Marion, D., Ikura, M., Tschudin, R. and Bax, A. (1989) J. Magn. Reson., 85, 393–399.
Mayo, K.H., Fan, F., Beavers, M.P., Eckardt, A., Keane, P., Hoekstra, W.J. and Andrade-Gordon, P. (1996) Biochim. Biophys. Acta, 1296, 95–102.
McDonnell, P.A., Shon, K., Kim, Y. and Opella, S.J. (1993) J. Mol. Biol., 233, 447–463.
Miller, H.I., Henzel, W.J., Ridgway, J.B., Kuang, W.-J., Chisholm, V. and Liu, C.-C. (1989) Biotechnology, 7, 698–704.
Milon, A., Miyazawa, T. and Higashijima, T. (1990) Biochemistry, 29, 65–75.
Mizutani, S., Mori, H., Shimazu, S., Sakaguchi, K. and Kobayashi, T. (1986) Biotechnol. Bioeng., 28, 204–209.
Mori, H., Yano, T., Kobayashi, T. and Shimizu, S. (1979) J. Chem. Eng. Japan, 12, 313–319.
Muhandiram, D.R. and Kay, L.E. (1994) J. Magn. Reson., B103, 203–216.
Neri, D., Wider, G. and Wüthrich, K. (1992a) FEBS Lett., 303, 129–135.
Neri, D., Wider, G. and Wüthrich, K. (1992b) Proc. Natl. Acad. Sci. USA, 89, 4397–4401.
Ni, F., Zhu, Y. and Scheraga, H.A. (1995) J. Mol. Biol., 252, 656–671.
Nilsson, B., Forsberg, G., Moks, T., Hartmanis, M. and Uhlén, M. (1992) Curr. Opin. Struct. Biol., 2, 569–575.
Okada, A., Wakamatsu, K., Miyazawa, T. and Higashijima, T. (1994) Biochemistry, 33, 9438–9446.
Özkaynak, E., Finley, D. and Varshavsky, A. (1984) Nature, 312, 663–666.
Piotto, M., Saudek, V. and Sklenář, V. (1992) J. Biomol. NMR, 2, 661–665.
Ripoll, D.R. and Ni, F. (1992) Biopolymers, 32, 359–365.
Schaffner, W. and Weissmann, C. (1973) Anal. Biochem., 56, 502–514.
Schägger, H. and von Jagow, G. (1987) Anal. Biochem., 166, 368–379.
Scherf, T., Hiller, R., Naider, F., Levitt, M. and Anglister, J. (1992) Biochemistry, 31, 6884–6897.
Schwalbe, H., Fiebig, K.M., Buck, M., Jones, J.A., Grimshaw, S.B., Spencer, A., Glaser, S.J., Smith, L.J. and Dobson, C.M. (1997) Biochemistry, 36, 8977–8991.
Shaka, A.J., Keeler, J. and Freeman, R. (1983) J. Magn. Reson., 53, 313–340.
Shaka, A.J., Barker, P.B. and Freeman, R. (1985) J. Magn. Reson., 64, 547–552.
Simcox, M.E., Huvar, A., Simcox, T.G. and Vega, Q. (1994) Strategies, 7, 68–69.
Studier, F.W., Rosenberg, A.H., Dunn, J.J. and Dubendorff, J.W. (1990) Methods Enzymol., 185, 60–89.
Suzuki, K., Shimoi, H., Iwasaki, Y., Kawahara, T., Matsuura, Y. and Nishikawa, Y. (1990) EMBO J., 9, 4259–4265.
Takahashi, K., Okamoto, H., Seino, H. and Noguchi, M. (1990) Biochem. Biophys. Res. Commun., 169, 524–530.
Van de Ven, F.J.M., van Os, J.W., Aelen, J.M., Wymenga, S.S., Remerowski, M.L., Konings, R.N. and Hilbers, C.W. (1993) Biochemistry, 32, 8322–8328.
Wakamatsu, K., Okada, A., Miyazawa, T., Masui, Y., Sakakibara, S. and Higashijima, T. (1987) Eur. J. Biochem., 163, 331–338.
Wang, Z., Jones, J.D., Rizo, J. and Gierasch, L.M. (1993) Biochemistry, 32, 13991–13999.
Williams, K.A., Farrow, N.A., Deber, C.M. and Kay, L.E. (1996) Biochemistry, 35, 5145–5157.
Wishart, D.S., Bigam, C.G., Holm, A., Hodges, R.S. and Sykes, B.D. (1995a) J. Biomol. NMR, 5, 67–81.
Wishart, D.S., Bigam, C.G., Yao, J., Abildgaard, F., Dyson, H.J., Oldfield, E., Markley, J.L. and Sykes, B.D. (1995b) J. Biomol. NMR, 6, 135–140.
Wittekind, M. and Mueller, L. (1993) J. Magn. Reson., B101, 201–205.
Yao, L.J. and Mayo, K.H. (1996) Biochem. J., 315, 161–170.
Zhu, G. and Bax, A. (1990) J. Magn. Reson., 90, 405–410.
Zhu, G. and Bax, A. (1992) J. Magn. Reson., 100, 202–207.
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Kohno, T., Kusunoki, H., Sato, K. et al. A new general method for the biosynthesis of stable isotope-enriched peptides using a decahistidine-tagged ubiquitin fusion system: An application to the production of mastoparan-X uniformly enriched with 15N and 15N/13C. J Biomol NMR 12, 109–121 (1998). https://doi.org/10.1023/A:1008254603368
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DOI: https://doi.org/10.1023/A:1008254603368