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dUTPase from Escherichia coli; high-level expression and one-step purification.
Rebecca Persson1, Johan Nord, Robert Roth
1Department of Chemistry, University of York, Heslington, UK. persson@ysbl.york.ac.uk
Preparative Biochemistry & Biotechnology
|June 20, 2002
Summary
Researchers enhanced the overexpression of Escherichia coli deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) using a new cloning strategy. This simplified purification to a single step, yielding highly active enzyme in greater quantities.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Deoxyuridine 5 -triphosphate nucleotidohydrolase (dUTPase) is crucial for DNA synthesis and repair.
- Overexpression and efficient purification of dUTPase are essential for biochemical and structural studies.
- Previous methods for dUTPase purification were complex and yielded less enzyme.
Purpose of the Study:
- To reclon the dut gene for enhanced overexpression of E. coli dUTPase.
- To develop a simplified, one-step purification protocol for recombinant dUTPase.
- To achieve a homogenous preparation of highly active dUTPase.
Main Methods:
- The dut gene was cloned into the pET-3a vector for expression in E. coli BL21(DE3) pLysS.
- Expression was driven by a bacteriophage T7 promoter with induction.
- One-step purification was achieved using phosphocellulose chromatography at low pH.
Main Results:
- Recombinant dUTPase constituted 60% of the total extracted protein after induction.
- A homogenous preparation of dUTPase with high specific activity was obtained.
- The yield of purified enzyme increased significantly to 500 mg per litre of bacterial culture.
Conclusions:
- The recloned dut gene facilitates high-level expression of E. coli dUTPase.
- A simplified one-step purification protocol yields a highly active and homogenous enzyme preparation.
- This improved method significantly enhances the yield of purified dUTPase, facilitating further research.