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A fast method for obtaining highly pure recombinant herpes simplex virus type 1 thymidine kinase
J Fetzer1, M Michael, T Bohner
1Department of Pharmacy, Swiss Federal Institute of Technology, ETH-Zentrum, Zürich.
Protein Expression and Purification
|October 1, 1994
Summary
Recombinant Herpes Simplex Virus Type 1 thymidine kinase (TK) was efficiently purified from E. coli using a two-step method. The resulting enzyme exhibits biochemical characteristics identical to native viral TK.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Herpes Simplex Virus Type 1 thymidine kinase (TK) is crucial for viral DNA replication.
- Efficient and gentle purification of recombinant TK is essential for biochemical studies.
Purpose of the Study:
- To develop a fast and gentle two-step purification procedure for recombinant Herpes Simplex Virus Type 1 thymidine kinase (TK).
- To characterize the biochemical properties of the purified recombinant TK.
Main Methods:
- Expression of TK as a glutathione S-acetyl transferase fusion protein in E. coli.
- Purification using glutathione affinity chromatography followed by thrombin cleavage.
- Second purification step utilizing ATP affinity chromatography.
- Analysis by SDS-PAGE, mass spectrometry, and N-terminal sequencing.
Main Results:
- A two-step purification yielded homogeneously pure recombinant TK.
- Two novel thrombin cleavage sites were identified.
- The purified enzyme demonstrated enzymatic activity with a Km of 0.2 microM for thymidine.
- Native molecular weight determination indicated the enzyme functions as a homodimer.
Conclusions:
- The developed purification method is effective for obtaining pure and active recombinant HSV-1 TK.
- The recombinant enzyme shares biochemical characteristics with the native viral TK.
- The findings support the use of this recombinant TK in further biochemical and structural studies.