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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
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
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
2007-11-08 14:33:17
date last changed
2017-01-01 07:01:29
@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},
  volume       = {32},
  year         = {2002},
}