Overnight Express™ Autoinduction NMR Medium-Optimization provides reagents to make 1 L of
chemically defined autoinducing liquid culture medium used to optimize cell growth and protein expression before preparing isotopically labeled proteins for NMR spectroscopy. The autoinduction method is based on the differential metabolism of carbon sources in the medium to promote growth to high cell density and to automatically induce protein expression under control of lac promoters, eliminating the need to monitor cell densities or add inducer (IPTG). The system includes four concentrated sterile solutions: OnEx NMR Solution 1 is a blend of carbon sources optimized for tightly regulated uninduced growth to high density, followed by induction; OnEx Solution 2 is a buffer and nitrogen blend that mediates metabolic acid production and provides additional nitrogen for increased protein synthesis; OnEx Solution 3 provides magnesium for maximal cell density; OnEx Solution 4 provides trace metals to minimize growth limitations associated with mineral deficiencies and to satisfy the requirements of metal-containing target proteins, even at high expression levels. The kit also includes unlabeled solid ammonium chloride and reagents to make a non-inducing starter medium, for staged growth without expression and for stable storage of clones. Addition of the four liquid components and a sterile ammonium chloride solution to sterile water results in a chemically- defined medium promoting high cell densities and autoinduction of expression. When the user provides and substitutes ¹⁵N-labeled ammonium chloride for the supplied unlabeled reagent, this kit can be used to prepare ¹⁵N-labeled proteins.