title: Refolding of recombinant Pneumocystis carinii dihydrofolate reductase and characterization of the enzyme.
abstract: 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.
authors.affiliation: Department of Molecular Sciences, Wellcome Research Laboratories, Kent, United Kingdom.
authors.forename: C J
authors.initials: CJ
authors.lastname: Delves
authors.forename: S P
authors.initials: SP
authors.lastname: Ballantine
authors.forename: R L
authors.initials: RL
authors.lastname: Tansik
authors.forename: D P
authors.initials: DP
authors.lastname: Baccanari
authors.forename: D K
authors.initials: DK
authors.lastname: Stammers
mesh_terms: 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
chemical_list: D005493:Folic Acid Antagonists; D011994:Recombinant Proteins; D009249:NADP; D004247:DNA; D013762:Tetrahydrofolate Dehydrogenase