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dUTPase from the Retrovirus Equine Infectious Anemia Virus: High-Level Expression in Escherichia coli and Purification

Bergman, Anna Carin; Björnberg, Olof LU ; Nord, Johan; Rosengren, Anna Maria and Nyman, Per-Olof LU (1995) In Protein Expression and Purification 6(3). p.379-387
Abstract
Deoxyuridine 5′-triphosphate nucleotidohydrolase (dUTPase, EC 3.6.1.23) catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate, and plays important roles in nucleotide metabolism and DNA replication. The dUTPase gene of the retrovirus equine infectious anemia virus (EIAV) was cloned and overexpressed in Escherichia coli using the T7 RNA polymerase expression system. The recombinant vector (pET-3a/EDU), constructed by mutagenic PCR, was transformed into E. coli BL21(DE3) pLysS cells, resulting in expression of EIAV dUTPase at about 40% of the extracted protein, This level of overproduction is very high compared to previous reports on heterologous expression of dUTPases in E. coli. A one-step purification procedure using phosphocellulose... (More)
Deoxyuridine 5′-triphosphate nucleotidohydrolase (dUTPase, EC 3.6.1.23) catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate, and plays important roles in nucleotide metabolism and DNA replication. The dUTPase gene of the retrovirus equine infectious anemia virus (EIAV) was cloned and overexpressed in Escherichia coli using the T7 RNA polymerase expression system. The recombinant vector (pET-3a/EDU), constructed by mutagenic PCR, was transformed into E. coli BL21(DE3) pLysS cells, resulting in expression of EIAV dUTPase at about 40% of the extracted protein, This level of overproduction is very high compared to previous reports on heterologous expression of dUTPases in E. coli. A one-step purification procedure using phosphocellulose chromatography results in a homogeneous preparation of the enzyme in a yield of 45 mg liter−1 of bacterial culture. The purified EIAV dUTPase, run on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis, shows an apparent molecular mass of 15.1 kDa in accordance with the gene structure. The isoelectric point (pI) was determined to 5.6. Gel filtration under nondenaturating conditions gives a retention volume corresponding to a molecular mass of 40.8 kDa, suggesting a trimeric organization of the enzyme. The amino acid composition and amino-terminal sequence of the recombinant dUTPase are in agreement with predictions from the DNA sequence. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Protein Expression and Purification
volume
6
issue
3
pages
379 - 387
publisher
Academic Press
external identifiers
  • scopus:0029310642
ISSN
1046-5928
DOI
10.1006/prep.1995.1050
language
English
LU publication?
yes
id
45b536e7-ea2e-473f-9e10-c78df088f923 (old id 126407)
date added to LUP
2007-07-04 12:04:57
date last changed
2017-08-06 03:42:18
@article{45b536e7-ea2e-473f-9e10-c78df088f923,
  abstract     = {Deoxyuridine 5′-triphosphate nucleotidohydrolase (dUTPase, EC 3.6.1.23) catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate, and plays important roles in nucleotide metabolism and DNA replication. The dUTPase gene of the retrovirus equine infectious anemia virus (EIAV) was cloned and overexpressed in Escherichia coli using the T7 RNA polymerase expression system. The recombinant vector (pET-3a/EDU), constructed by mutagenic PCR, was transformed into E. coli BL21(DE3) pLysS cells, resulting in expression of EIAV dUTPase at about 40% of the extracted protein, This level of overproduction is very high compared to previous reports on heterologous expression of dUTPases in E. coli. A one-step purification procedure using phosphocellulose chromatography results in a homogeneous preparation of the enzyme in a yield of 45 mg liter−1 of bacterial culture. The purified EIAV dUTPase, run on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis, shows an apparent molecular mass of 15.1 kDa in accordance with the gene structure. The isoelectric point (pI) was determined to 5.6. Gel filtration under nondenaturating conditions gives a retention volume corresponding to a molecular mass of 40.8 kDa, suggesting a trimeric organization of the enzyme. The amino acid composition and amino-terminal sequence of the recombinant dUTPase are in agreement with predictions from the DNA sequence.},
  author       = {Bergman, Anna Carin and Björnberg, Olof and Nord, Johan and Rosengren, Anna Maria and Nyman, Per-Olof},
  issn         = {1046-5928},
  language     = {eng},
  number       = {3},
  pages        = {379--387},
  publisher    = {Academic Press},
  series       = {Protein Expression and Purification},
  title        = {dUTPase from the Retrovirus Equine Infectious Anemia Virus: High-Level Expression in Escherichia coli and Purification},
  url          = {http://dx.doi.org/10.1006/prep.1995.1050},
  volume       = {6},
  year         = {1995},
}