Cloning, expression, and characterization of Cryptococcus neoformans dihydrofolate reductase.
The Cryptococcus neoformans dihydrofolate reductase (DHFR) gene has been isolated from cDNA and genomic DNA libraries. The 690-base pair coding sequence codes for a 25,152-Da protein, which is the largest monofunctional DHFR yet reported. The gene contains two introns, and several putative regulatory sequences have been identified. The coding sequence was placed in a pUC-based expression vector, which expresses C. neoformans DHFR in Escherichia coli at a level of about 5% of the total soluble extract. The expressed DHFR was purified to homogeneity by methotrexate-Sepharose affinity chromatography, followed by anion exchange chromatography on Q-Sepharose. On SDS-polyacrylamide gel electrophoresis, the purified enzyme migrates as a single protein with apparent mass of 28 kDa. The molecular weight, as determined by electrospray mass spectral analysis, and the amino-terminal sequence are in accord with what was predicted from the DNA sequence. Steady state kinetic parameters, effects of pH, salts, and inhibition constants of several anti-folates have been determined.[1]References
- Cloning, expression, and characterization of Cryptococcus neoformans dihydrofolate reductase. Sirawaraporn, W., Cao, M., Santi, D.V., Edman, J.C. J. Biol. Chem. (1993) [Pubmed]
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