CLONING, EXPRESSION AND CHARACTERIZATION OF THYMIDYLATE SYNTHASE FROM CRYPTOCOCCUS-NEOFORMANS

The thymidylate synthase (TS)-encoding gene from Cryptococcus neoformans (Cn) has been isolated from cDNA and genomic libraries. The 1127-bp gene contains three introns and a 951-bp open reading frame encoding a 35 844-Da protein. The cDNA clones lack 324 bp of the 5' coding region of the gene, The complete coding sequence was assembled as an expression cassette in pUC19 using parts of the coding sequence from the cDNA and genomic DNA and completing the sequence using synthetic DNA. Production of active TS from Cn (CnTS) was first demonstrated by complementation of a thymine(Thy)-requiring Escherichia coli strain. The expression cassette was subsequently subcloned into the T7 polymerase vector pET15-b. In this construct, CnTS is produced as approximately 10% of the total soluble protein in E. coli. Homogeneous enzyme was obtained at a 36% yield after consecutive chromatography on DEAE-cellulose, Q-Sepharose, phenyl-Sepharose and Affi-Gel Blue. Steady-state kinetic analysis showed that the K-m values for dUMP and CH2H4.folate were 2.7+/-0,5 mu M and 38.2+/-2.5 mu M, respectively, and the k(cat) was 5.1 s(-1). The enzyme was stable upon storage at -80 degrees C in Tris.HCl pH 7.4 and thiol.