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

Persson, R ; Nord, J ; Roth, Robert LU and Nyman, Per-Olof LU (2002) In Preparative Biochemistry & Biotechnology 32(2). p.157-172
Abstract
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... (More)
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. (Less)
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author
; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Preparative Biochemistry & Biotechnology
volume
32
issue
2
pages
157 - 172
publisher
Taylor & Francis
external identifiers
  • pmid:12071646
  • wos:000176220200006
  • scopus:0035985130
  • pmid:12071646
ISSN
1532-2297
DOI
10.1081/PB-120004128
language
English
LU publication?
yes
id
b5a95ed3-560a-41bf-898d-8b5452b6b3c1 (old id 335409)
date added to LUP
2016-04-01 16:18:10
date last changed
2022-02-27 20:14:22
@article{b5a95ed3-560a-41bf-898d-8b5452b6b3c1,
  abstract     = {{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.}},
  author       = {{Persson, R and Nord, J and Roth, Robert and Nyman, Per-Olof}},
  issn         = {{1532-2297}},
  language     = {{eng}},
  number       = {{2}},
  pages        = {{157--172}},
  publisher    = {{Taylor & Francis}},
  series       = {{Preparative Biochemistry & Biotechnology}},
  title        = {{dUTPase from Escherichia coli; high-level expression and one-step purification}},
  url          = {{http://dx.doi.org/10.1081/PB-120004128}},
  doi          = {{10.1081/PB-120004128}},
  volume       = {{32}},
  year         = {{2002}},
}