CLONING, EXPRESSION, AND CHARACTERIZATION OF CRYPTOCOCCUS-NEOFORMANS DIHYDROFOLATE-REDUCTASE

The Cryptococcus neoformans dihydrofolate reductase (DHFR) gene has be en isolated from cDNA and genomic DNA libraries. The 690-base pair cod ing sequence codes for a 25,152-Da protein, which is the largest monof unctional DHFR yet reported. The gene contains two introns, and severa l putative regulatory sequences have been identified. The coding seque nce was placed in a pUC-based expression vector, which expresses C. ne oformans DHFR in Escherichia coli at a level of about 5% of the total soluble extract. The expressed DHFR was purified to homogeneity by met hotrexate-Sepharose affinity chromatography, followed by anion exchang e chromatography on Q-Sepharose. On SDS-polyacrylamide gel electrophor esis, the purified enzyme migrates as a single protein with apparent m ass of 28 kDa. The molecular weight, as determined by electrospray mas s spectral analysis, and the amino-terminal sequence are in accord wit h what was predicted from the DNA sequence. Steady state kinetic param eters, effects of pH, salts, and inhibition constants of several anti- folates have been determined.