Refolding of recombinant Pneumocystis carinii dihydrofolate reductase and characterization of the enzyme.
The isolation of dihydrofolate reductase (DHFR) cDNA sequences from the messenger RNA of Pneumocystis carinii using the polymerase chain reaction is described. The 206-amino acid P. carinii DHFR was expressed to high levels in Escherichia coli inclusion bodies using the T7 promoter expression system. Solubilization of the inclusion bodies in 4 M guanidine hydrochloride and refolding of the recombinant protein in the presence of 0.5% polyethylene glycol 1450 yielded correctly folded DHFR which was purified to homogeneity by methotrexate-Sepharose affinity chromatography. The refolded enzyme was readily crystallized as a ternary complex with NADPH and various inhibitors. The enzyme exhibited a sharp pH optimum with maximum activity at pH 7.0 (turnover number = 6500 min-1). Km values for dihydrofolate (DHF) and NADPH were 2.3 and 3.0 microM, respectively, in 0.1 m imidazole buffer, pH 7. Folate did not act as a substrate. Comparison of the kinetic properties of the refolded enzyme with soluble P. carinii DHFR expressed at low levels in the T7 expression system showed similar pH-activity profiles, Km values for DHF and NADPH, and IC50 values for several known antifolates which were tested as inhibitors of the enzyme.
Delves
C J
CJ
Department of Molecular Sciences, Wellcome Research Laboratories, Kent, United Kingdom.
Ballantine
S P
SP
Tansik
R L
RL
Baccanari
D P
DP
Stammers
D K
DK
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA; D004926:Escherichia coli; D005493:Folic Acid Antagonists; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D011010:Pneumocystis / Q000201:enzymology*; D016133:Polymerase Chain Reaction; D017510:Protein Folding; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D005493:Folic Acid Antagonists; D011994:Recombinant Proteins; D009249:NADP; D004247:DNA; D013762:Tetrahydrofolate Dehydrogenase