| Article ID | Journal | Published Year | Pages | File Type |
|---|---|---|---|---|
| 2021463 | Protein Expression and Purification | 2007 | 8 Pages |
The full length human adenylosuccinate lyase gene was generated by a PCR method using a plasmid encoding a truncated human enzyme as template, and was cloned into a pET-14b vector. Human adenylosuccinate lyase was overexpressed in Escherichia coli Rosetta 2(DE3)pLysS as an N-terminal histidine-tagged protein and was purified to homogeneity by a nickel–nitriloacetic acid column at room temperature. The histidine tag was removed from the human enzyme by thrombin digestion and the adenylosuccinate lyase was purified by Sephadex G-100 gel filtration. The histidine-tagged and non-tagged adenylosuccinate lyases exhibit similar values of Vmax and Km for S-AMP. Analytical ultracentrifugation and circular dichroism revealed, respectively, that the histidine-tagged enzyme is in tetrameric form with a molecular weight of 220 kDa and contains predominantly α-helical structure. This is the first purification procedure to yield a stable form of human adenylosuccinate lyase. The enzyme is stable for at least 5 days at 25 °C, and upon rapid freezing and thawing. Temperature as well as reducing agent (DTT) play critical roles in determining the stability of the human adenylosuccinate lyase.
