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Cloning, expression and characterization of thymidylate synthase from Cryptococcus neoformans
L L Livi1, U Edman, G P Schneider
1Department of Pharmaceutical Chemistry, University of California, San Francisco 94143-0448.
Gene
|December 15, 1994
Summary
The thymidylate synthase (TS) gene from Cryptococcus neoformans was cloned and expressed in E. coli. This study details the successful production and purification of active CnTS, providing insights into its kinetic properties.
Area of Science:
- Molecular Biology
- Enzymology
- Mycology
Background:
- Thymidylate synthase (TS) is a crucial enzyme in nucleotide biosynthesis.
- Understanding fungal TS can lead to targeted antifungal therapies.
- Cryptococcus neoformans is an important human fungal pathogen.
Purpose of the Study:
- To isolate and characterize the thymidylate synthase (TS) gene from Cryptococcus neoformans (Cn).
- To produce and purify active CnTS for further study.
- To determine the kinetic parameters of the purified enzyme.
Main Methods:
- Gene isolation from cDNA and genomic libraries.
- Construction of an expression cassette for recombinant protein production in E. coli.
- Purification using multiple chromatography techniques (DEAE-cellulose, Q-Sepharose, phenyl-Sepharose, Affi-Gel Blue).
- Enzyme activity assays and kinetic analysis.
Main Results:
- The complete coding sequence of CnTS was assembled and expressed in E. coli.
- Active CnTS was produced, representing approximately 10% of total soluble protein.
- Homogeneous enzyme was obtained with a 36% yield.
- Kinetic parameters (Km for dUMP and CH2H4.folate, kcat) were determined.
Conclusions:
- The gene encoding thymidylate synthase from Cryptococcus neoformans has been successfully cloned, expressed, and purified.
- The characterized enzyme exhibits stable properties and defined kinetic parameters.
- This work provides a foundation for further investigation of fungal TS and potential drug development.