Protocols have been developed and applied for the high-throughput production of [U-N-15]- or [U-C-13-, U-15 N]-labeled proteins using the conditional methionine auxotroph Escherichia coli B834. The large-scale growth and expression uses a chemically defined auto-induction medium containing salts and trace metals, vitamins including vitamin B-12,, and glucose, glycerol, and lactose. The results from nine expression trials in 2-L of the auto-induction medium (500 mL in each of four polyethylene terephthalate beverage bottles) gave an average final optical density at 600 nm of similar to 5, an average wet cell mass yield of -9.5 g L-1, and an average yield of similar to 20 mg of labeled protein in the six instances in which proteolysis of the fusion protein was observed. Correlations between the cell mass recovered, the level of protein expression, and the relative amounts of glucose, glycerol, and lactose in the auto-induction medium were noted. Mass spectral analysis showed that the purified proteins contained both N-15 and C-13 at levels greater than 95%. H-1-N-15 heteronuclear single quantum correlation spectroscopy as well as, C-13; N-15-edited spectroscopy showed that the purified [U-(15) N]- and [U-C-13, U-(15) N]-labeled proteins were suitable for structure analysis. (c) 2005 Elsevier Inc. All rights reserved.