Isolation and expression of the Pneumocystis carinii thymidylate synthase gene

Abstract

The thymidylate synthase (TS) gene from Pneumocystis carinii has been isolated from complementary and genomic DNA libraries and expressed in Escherichia coli. The coding sequence of TS is 891 nucleotides, encoding a 297-amino acid protein of Mr 34,269. The deduced amino acid sequence is similar to TS from other organisms and is most closely related to the enzyme from Saccharomyces cerevisiae with 65% identity. TS is found on a 330-kilobase-pair chromosome in P. carinii. While TS and dihydrofolate reductase reside on a single polypeptide chain in all protozoa studied to date, TS is not linked to dihydrofolate reductase in P. carinii. The TS gene shows the presence of four small intervening sequences, some of which interrupt the coding sequence in highly ordered structural regions of the protein. Heterologous expression of P. carinii TS in E. coli was accomplished by cloning the coding sequence into plasmid vectors under control of the lac and tac promoters. These constructs direct the synthesis of catalytically active enzyme to the extent of 2% of total soluble protein.