Related Experiment Videos
Recombinant human cytidine deaminase: expression, purification, and characterization
S Vincenzetti1, A Cambi, J Neuhard
1Dipartimento di Biologia M.C.A., Università di Camerino, via Camerini, 3, Camerino (MC), 62032, Italy.
Protein Expression and Purification
|September 1, 1996
Summary
Researchers cloned human cytidine deaminase (CDA) and expressed it in E. coli. The purified recombinant CDA enzyme is a tetramer, contains zinc, and exhibits high activity, crucial for understanding antitumoral drug metabolism.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Human cytidine deaminase (CDA) plays a role in drug metabolism.
- Understanding CDA's structure and function is vital for developing antitumoral therapies.
Purpose of the Study:
- To clone and express human CDA in Escherichia coli.
- To characterize the recombinant human CDA enzyme's properties and activity.
Main Methods:
- Reverse transcriptase PCR was used to obtain CDA cDNA from peripheral blood leukocytes.
- The cDNA was ligated into an expression vector for bacterial expression.
- Recombinant CDA was purified using heat inactivation and affinity chromatography.
Main Results:
- The expressed protein showed high CDA activity and was identified as a tetramer.
- Purified recombinant CDA had a specific activity of 105 U/mg and was >98% pure.
- ICP-OES analysis confirmed the presence of one zinc atom per subunit.
Conclusions:
- Recombinant human CDA was successfully expressed, purified, and characterized.
- The enzyme's tetrameric structure and zinc cofactor are confirmed.
- This work provides a valuable resource for studying CDA's role in drug deamination and metabolism.