Cloning and expression in Escherichia coli of the Clostridium thermoaceticum gene encoding thermostable formyltetrahydrofolate synthetase (original) (raw)
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 (μmol·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.
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Abbreviations
FTHFS:
formyltetrahydrofolate synthetase
kb:
kilobase
H4-folate:
tetrahydrofolate
SDS:
sodium dodecyl sulfate
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- Charles R. Lovell
Present address: Department of Biology, University of South Carolina, 29208, Columbia, SC, USA
Authors and Affiliations
- Department of Biochemistry, University of Georgia, 30602, Athens, GA, USA
Charles R. Lovell, Alan Przybyla & Lars G. Ljungdahl - Center for Biological Resource Recovery, University of Georgia, 30602, Athens, GA, USA
Charles R. Lovell, Alan Przybyla & Lars G. Ljungdahl
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- Charles R. Lovell
You can also search for this author inPubMed Google Scholar - Alan Przybyla
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Lovell, C.R., Przybyla, A. & Ljungdahl, L.G. Cloning and expression in Escherichia coli of the Clostridium thermoaceticum gene encoding thermostable formyltetrahydrofolate synthetase.Arch Microbiol 149, 280–285 (1988). https://doi.org/10.1007/BF00411642
- Received: 06 August 1987
- Accepted: 26 October 1987
- Issue Date: February 1988
- DOI: https://doi.org/10.1007/BF00411642