dUTPase from Escherichia coli; high-level expression and one-step purification
(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)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/335409
- author
- Persson, R ; Nord, J ; Roth, Robert LU and Nyman, Per-Olof LU
- organization
- publishing date
- 2002
- 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}}, }