STABILITY OF MUTANT TYPE-II DIHYDROFOLATE-REDUCTASE PROTEINS IN SUPPRESSOR STRAINS

In the course of study of a (Glu-58 to Gln-58) mutant type II dihydrofolate reductase (DHFR), it was found that the altered DHFR was poorly produced in vivo. Investigations with several common laboratory Escherichia coli strains including htpR and lon strains bearing plasmids expressing the Gln-58 DHFR indicated a correlation of rapid degradation with the presence of a sup+ phenotype. The sup-degrees strain MC1061(p3) was transformed with a series of plasmids containing the Gln-58 DHFR gene with an without an additional supF gene, and expression levels were compared. The supF+ constructs exhibited little accumulation of the Gln-58 DHFR, while reasonable levels were found in the sup-degrees cases. Experiments with extracts of plasmid-free sup+ and sup-degrees strains showed rapid degradation by certain strains compared to MC1061(p3) and this degradation was not dependent upon ATP. In another route to increasing the stability of labile DHFR derivatives, mutagenesis of a strain bearing a N-terminally shortened Gln-58 DHFR was performed. Selection and analysis of a trimethoprim-resistant stable mutant showed that this DHFR gene contained a triple repeat of leu-pro-ser in the enzymatically non-essential N-terminal portion of the protein.