A novel protocol for isotopically labeling bacterially expressed proteins is presented. This method circumvents problems related to poor cell growth, commonly associated with the use of minimal labeled media, and problems with protein induction encountered, less commonly, when using enriched labeled media. The method involves initially growing the bacterial cells to high optical density in a commercially available enriched labeled medium. Following a suitable growth period, the cells are transferred to a different (minireal) labeled medium, appropriate for induction. The method is demonstrated using the protein melanoma growth stimulating activity (MGSA).
Recent advances in NMR spectroscopy have made it possible to determine the solution conformations of medium-sized proteins up to molecular masses of about 30 kDa (Clore and Gronenborn, 1991a,b;Bax and Grzesiek, 1993;Wagner, 1993). The new methods involve implementation of a number of 3D and 4D heteronnclear NMR techniques and require that the protein of interest be uniformly labeled with the NMR-observable isotopes 15N and/or 13C. Uniform isotopic labeling of proteins is most commonly achieved by expressing the protein in a microbial host, typically Escherichia coli, grown in a minimal medium in which the only nitrogen source is 'SN-labeled (e.g. (15NH4)~SO4 or 15NH4C1) and the only carbon source is 13C-labeled (e.g. 13C6-glucose ) (McIntosh and Dahlquist, 1990). Economic considerations require that the expression system be reasonably efficient under these growth conditions. Generally, however, bacteria grow and secrete more efficiently in enriched or supplemented media, rather than minimal media. An alternative approach to the use of 15N-and/or 13C-labeled minimal media therefore is to use commercially available media, composed of uniformly lSN-and/or 13C-enriched hydrolysates extracted from algae, grown with simple isotopically labeled raw materials (e.g. K~SNO2, and ~3CO2).
In some circumstances, however, neither of the above approaches will yield amounts of uni-*To whom correspondence should be addressed.