Cloning and expression in Escherichia coli of the Clostridium thermoaceticum gene encoding thermostable formyltetrahydrofolate synthetase.
Formyltetrahydrofolate synthetase (FTHFS) (EC 6.3.4.3), a thermostable protein of four identical subunits from Clostridium thermoaceticum was cloned into Escherichia coli SK1592. The clone (CRL47) contained a 9.5 kb EcoRI fragment of C. thermoaceticum DNA ligated into pBR322. It produced catalytically active, thermostable FTHFS, that was not found in E. coli SK1592 containing native pBR322. The identity of the expressed enzyme was confirmed by specific binding of rabbit polyclonal anti-FTHFS serum produced against C. thermoaceticum FTHFS. The specific activities (mumol.min-1.mg-1) of FTHFS in cell free extracts of CRL47 were 28-89 when assayed at 50 degrees C and pH 8. This was from 3-10-fold higher than in C. thermoaceticum extracts. FTHFS was purified to homogeneity from CRL47. The purified enzyme behaved during electrophoresis and gel chromatography and it had similar specific activity and thermostability as the enzyme purified from C. thermoaceticum.
Lovell
C R
CR
Department of Biochemistry, University of Georgia, Athens 30602.
Przybyla
A
A
Ljungdahl
L G
LG
D003001:Cloning, Molecular; D003013:Clostridium / Q000201:enzymology / Q000235:genetics*; D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial / Q000032:analysis; D003720:Densitometry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D005574:Formate-Tetrahydrofolate Ligase / Q000235:genetics*; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D007118:Immunoassay; D008025:Ligases / Q000235:genetics*; D009693:Nucleic Acid Hybridization
D004269:DNA, Bacterial; D004262:DNA Restriction Enzymes; D008025:Ligases; D005574:Formate-Tetrahydrofolate Ligase