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.
Sirawaraporn
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Hormone Research Institute, University of California, San Francisco 94143.
Cao
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Santi
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Edman
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JC
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003455:Cryptococcus neoformans / Q000201:enzymology* / Q000235:genetics; D004271:DNA, Fungal / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005493:Folic Acid Antagonists; D006863:Hydrogen-Ion Concentration; D007438:Introns; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins; D017386:Sequence Homology, Amino Acid; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D004271:DNA, Fungal; D005493:Folic Acid Antagonists; D011994:Recombinant Proteins; D013762:Tetrahydrofolate Dehydrogenase