한국자기공명학회논문지 (Journal of the Korean Magnetic Resonance Society)

한국자기공명학회 (Korean Magnetic Resonance Society)
권호 표지
DOI QR코드

DOI QR Code

Cost-effective isotope labeling technique developed for 15N/13C-labeled proteins

  • Kim, Hee-Youn (Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University) ;
  • Hong, Eun-Mi (Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University) ;
  • Lee, Weon-Tae (Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University)
  • 투고 : 2011.11.09
  • 심사 : 2011.12.13
  • 발행 : 2011.12.20
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

A newly developed cost-effective approach to prepare $^{15}N/^{13}C$-labeled protein for NMR studies is presented. This method has been successfully applied to isotopically labeling of PTK6 SH2 domain and MTH 1880 protein. The production method generates cell density using a growing media containing $^{15}NH_4Cl$, $^{12}C_6$-D-glucose. Following a doubling time period for unlabeled metabolite exhaustion and then addition $^{13}C_6$-D-glucose into a M9 growing media, the cells are induced. Our results demonstrate that in order to get full incorporation of $^{13}C$, the isotopes are not totally required during the initial growth phase before induction. The addition of small amounts of $^{13}C_6$-D-glucose to the induction phase is sufficient to obtain more than 95% incorporation of isotopes into the protein. Our optimized protocol is two-thirds less costly than the classical method using $^{13}C$ isotope during the entire growth phase.

키워드

참고문헌

  1. A.G. Palmer, C. Bracken, In NMR Supramolecular Chemistry (pons, M., Ed.). Kluwer Academic Publishers, Dordrecht, pp.171, (1999).
  2. W. Shu, Ji, H. Liu, M. Lu, J. Mol. Biol. 299. 1101, (2000). https://doi.org/10.1006/jmbi.2000.3776
  3. G.T. Montelione, D. Zheng, Y.J. Huang, Gunsalus, K.C. and Szyperski, T. Nat. Struct. Biol. 7, 982, (2000). https://doi.org/10.1038/80768
  4. A. Bax, S. Grzesiek, Acc. Chem. Res. 26, 131, (1993). https://doi.org/10.1021/ar00028a001
  5. G. M. Clore, A. M. Gronenborn, Science 252, 1390, (1991a). https://doi.org/10.1126/science.2047852
  6. G. M. Clore, A. M. Gronenborn, Prog. Nucl. Magn. Reson. Spectrosc. 23, 43, (1991b) https://doi.org/10.1016/0079-6565(91)80002-J
  7. G. M. Clore, A. M. Gronenborn, Methods Enzymol. 239, 349, (1994). https://doi.org/10.1016/S0076-6879(94)39013-4
  8. D. Reilly, W.J. Fairbrother, J. Biomol. NMR 4, 459, (1994).
  9. M. Jansson, Y.C. Li, S. Anderson, G. Montelione, B. Nilsson, J. Biomol. NMR 7, 131, (1996).
  10. M. Kainosho, Nat. Struct. Biol. 4, 858, (1997).
  11. M. Cai, H. Ying, K. Sakaguchi, G.M. Clore, A.M. Gronenborn, R. Craigie, J. Biomol. NMR 11, 97, (1998). https://doi.org/10.1023/A:1008222131470
  12. J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning : A Laboratory Manual. 3. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. (1989).
  13. E. Hong, J Shin, E. Bang, MH. Kim, ST, Lee, W. Lee J. Biomol. NMR 19, 291, (2001) https://doi.org/10.1023/A:1011221125013
  14. S. Grzesiek, A. Bax, J. Magn. Reson. 96, 432, (1992) https://doi.org/10.1016/0022-2364(92)90099-S
  15. L.E. Kay, G.Y. Xu, T. Yamazaki, J. Magn. Reson. A109, 129, (1994)
  16. J. Stonehouse, R.T. Clowes, G.L. Shaw, J. Keeler, E.D. Laue, J. Biomol. NMR 5, 226, (1995)
  17. D.R. Muhandiram, L.E. Kay, J. Magn. Reson. B103, 203, (1994)
  18. S. Grzesiek, A. Bax, J. Magn. Reson. 99, 201, (1992) https://doi.org/10.1016/0022-2364(92)90169-8
  19. M. Ikura, L.E. Kay, A. Bax, Biochemisty 29, 4659, (1990) https://doi.org/10.1021/bi00471a022
  20. A. Grzesiek, and A. Bax, J. Magn. Reson. B102, 103, (1993)
  21. L.E. Kay, G.Y. Xu, A.U. Singer, D.R. Muhandiram, J.D. Forman-Kay J. Magn. Reson. B101, 333, (1993)
  22. A.L. Davis, J. Keeler, E.D. Laue, D. J. Moskau, Magn. Reson. 98, 207, (1992) https://doi.org/10.1016/0022-2364(92)90126-R

피인용 문헌

  1. A new approach to quantify paraquat intoxication from postmortem blood sample by using1H qNMR method vol.17, pp.1, 2013, https://doi.org/10.6564/JKMRS.2013.17.1.040