Purification and characterization of recombinant Pneumocystis carinii thymidylate synthase.
Catalytically active Pneumocystis carinii thymidylate synthase is expressed to the extent of about 4% of the soluble protein in Escherichia coli chi 2913 harboring plasmid pUETS-1.8 (U. Edman, J. C. Edman, B. Lundgren, and D. V. Santi, Proc. Natl. Acad. Sci. USA 86, 6503-6507, 1989). Ion-exchange, affinity, hydrophobic, and reactive dye agarose chromatography steps were explored to devise a large-scale purification protocol for P. carinii thymidylate synthase. Sequential DE52, Q-Sepharose, phenyl-Sepharose, and Cibacron Blue F3GA chromatography yielded enzyme that was homogeneous by SDS-PAGE in a yield of over 50%. The sequence of the first 10 amino acid residues of the purified protein was in accord with that predicted from the DNA sequence. Isoelectric focusing gave a pI of 6.2. Kinetic analysis of the purified enzyme revealed that the Km values were 4.7 +/- 1.3 microM for dUMP and 15.7 +/- 4.3 microM for 5,10-methylenetetrahydrofolate, similar to those of many other thymidylate synthases; the kcat of the most active preparation was 0.8 s-1. The enzyme is stable for at least 2 months when stored at -80 degrees C in the presence of 40% glycerol, Tris-HCl, and thiol.
Santi
D V
DV
Department of Biochemistry and Biophysics, University of California, San Francisco 94143.
Edman
U
U
Minkin
S
S
Greene
P J
PJ
D000595:Amino Acid Sequence; D002845:Chromatography / Q000379:methods; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007526:Isoelectric Point; D007700:Kinetics; D008969:Molecular Sequence Data; D011010:Pneumocystis / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013940:Thymidylate Synthase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*
D011994:Recombinant Proteins; D013940:Thymidylate Synthase