dUTPase from Escherichia coli; high-level expression and one-step purification

• The dut gene, which encodes Eseherichia coli deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase), has been recloned to increase overexpression of the enzyme and to enable simplification of the purification protocol into a one-step procedure. The gene was cloned into the vector pET-3a and expressed in E. coli BL21(DE3) pLysS under the control of a bacteriophage T7 promotor. Induction results in production of dUTPase corresponding to 60% of the extracted protein. Phosphocellulose chromatography at low pH was utilised for one-step purification, resulting in a homogenous preparation of the recombinant protein with a highly specific activity. The yield of purified enzyme is 500mg per litre of bacterial culture, a significant increase compared to previously employed methods.

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NATURVETENSKAP  -- Biologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences (hsv//eng)

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