HIGH-LEVEL EXPRESSION AND ISOTOPIC LABELING OF LACTOBACILLUS-CASEI DIHYDROFOLATE-REDUCTASE FOR NUCLEAR-MAGNETIC-RESONANCE SPECTROSCOPY

Two efficient systems have been used for high-level expression of lact obacillus casei dihydrofolate reductase in Escherichia coli, including the production of protein generally and specifically labeled with C-1 3 and N-15. A system based on T7 RNA polymerase led to the production of dihydrofolate reductase at a level of 37% of the total soluble prot ein of the host strain: 50 mg of pure enzyme was obtained from a 1 lit er of culture (or 14 mg/g wet weight of cells). In this system, a smal l amount of the enzyme (less than 5%) was identified as a catalyticall y active 21-kDa fusion protein. Introduction of a second in-frame (och re) stop codon did not eliminate the production of this fusion protein . The same expression system was also used to prepare dihydrofolate re ductase generally labeled with N-15 and to prepare single and double m utants of the enzyme. In order to have an expression system which can be used with a range of auxotrophic strains of E. coli, a system based on the tac promoter was used. This led to the production of dihydrofo late reductase at a level of 29% of total soluble protein; a yield of 40 mg enzyme per liter of culture (or 11 mg/g wet weight of cells). Th is system was successfully used to produce mutants of the enzyme as we ll as the enzyme selectively labeled with [gamma-C-13]aspartic acid. ( C) 1995 Academic Press, Inc.