An efficient and cost-effective isotope labeling protocol for proteins expressed in shape Escherichia coli
- 440 Downloads
A cost-effective protocol for uniform 15N and/or13 C isotope labeling of bacterially expressed proteins is presented. Unlike most standard protocols, cells are initially grown in a medium containing nutrients at natural abundance and isotopically labeled nutrients are only supplied at the later stages of growth and during protein expression. This permits the accumulation of a large cell mass without the need to employ expensive isotopically labeled nutrients. The abrupt decrease in oxygen consumption that occurs upon complete exhaustion of essential nutrients is used to precisely time the switch between unlabeled and labeled nutrients. Application of the protocol is demonstrated for wild-type and a mutant of the N-terminal zinc-binding domain of HIV-1 integrase.
Unable to display preview. Download preview PDF.
- Bax, A. and Grzesiek, S. (1993) Acc. Chem. Res., 26, 131–138.Google Scholar
- Cai, M., Zheng, R., Caffrey, M., Craigie, R., Clore, G.M. and Gronenborn, A.M. (1997) Nat. Struct. Biol., 4, 567–577.Google Scholar
- Clore, G.M. and Gronenborn, A.M. (1991a) Science, 252, 1390–1399.Google Scholar
- Clore, G.M. and Gronenborn, A.M.(1991b) Prog. Nucl. Magn. Reson. Spectrosc., 23, 43–92.Google Scholar
- Muchmore, D.C., McIntosh, L.P., Russel, C.B., Anderson, D.E. and Dahlquist, F.W. (1989) Methods Enzymol., 177, 44–73.Google Scholar
- Reilly, D. and Fairbrother, W.J.(1994) J. Biomol. NMR, 4, 459–462.Google Scholar
- Studier, F.W., Rosenberg, A.H., Dunn, J.J., and Dubendorf, J.W. (1990)Methods Enzymol., 185, 60–89.Google Scholar
- Venters, R.A., Calderone, T.L., Spicer, L.D. and Fierke, C.A. (1991)Biochemistry, 30, 4491–1194.Google Scholar