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 lactobacillus casei dihydrofolate reductase in Escherichia coli, including the production of protein generally and specifically labeled with C-13 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 protein of the host strain: 50 mg of pure enzyme was obtained from a 1 liter of culture (or 14 mg/g wet weight of cells). In this system, a small amount of the enzyme (less than 5%) was identified as a catalytically active 21-kDa fusion protein. Introduction of a second in-frame (ochre) stop codon did not eliminate the production of this fusion protein. The same expression system was also used to prepare dihydrofolate reductase generally labeled with N-15 and to prepare single and double mutants 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 dihydrofolate 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). This system was successfully used to produce mutants of the enzyme as well as the enzyme selectively labeled with [gamma-C-13]aspartic acid. (C) 1995 Academic Press, Inc.