Cloning and expression in Escherichia coli of the Clostridium thermoaceticum gene encoding thermostable formyltetrahydrofolate synthetase |
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Authors: | Charles R Lovell Alan Przybyla Lars G Ljungdahl |
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Institution: | (1) Department of Biochemistry, University of Georgia, 30602 Athens, GA, USA;(2) Center for Biological Resource Recovery, University of Georgia, 30602 Athens, GA, USA;(3) Present address: Department of Biology, University of South Carolina, 29208 Columbia, SC, USA |
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Abstract: | 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 ( mol·min-1·mg-1) of FTHFS in cell free extracts of CRL47 were 28–89 when assayed at 50°C and pH8. 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.Abbreviations FTHFS
formyltetrahydrofolate synthetase
- kb
kilobase
- H4-folate
tetrahydrofolate
- SDS
sodium dodecyl sulfate
A preliminary account of this work was presented at the annual meeting of the American Society for Microbiology, Atlanta, GA, 1987 (C. R. Lovell, A. Przybyla and L. G. Ljungdahl, Abstr. Annu. Meet. Am. Soc. Microbiol. 1987, K126, p. 223). |
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Keywords: | Clostridium thermoaceticum Formyltetrahydrofolate synthetase Cloning Escherichia coli Thermostable protein Expression Acetogen |
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