High-level expression of enzymatically active bovine leukemia virus proteinase in E. coli

Martin Andreansky, Olga Hrušková-Heidingsfeldová, Juraj Sedláčeka, Jan Konvalinka, Ivo Bláha, Petr Ječmen, Magda Hořejši, Petr Štrop, Milan Fábry

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

An E. coli plasmid expressing efficiently an artificial precursor of bovine leukemia virus (BLV) proteinase under transcriptional control of the phage T7 promoter was constructed. The expression product accumulates in the induced E. coli cells in the form of insoluble cytoplasmic inclusions. Solubilization of the inclusions and a refolding step yield almost pure and completely self-processed proteinase. Purification to homogeneity was achieved by ion-exchange chromatography and reverse-phase HPLC. On a preparative scale, a high yield of enzymatically active proteinase was obtained. An initial study using a series of synthetic peptide substrates shows a distinct substrate specificity of BLV proteinase.

Original languageEnglish
Pages (from-to)129-132
Number of pages4
JournalFEBS Letters
Volume287
Issue number1-2
DOIs
StatePublished - Aug 5 1991
Externally publishedYes

Fingerprint

Bovine Leukemia Virus
Viruses
Escherichia coli
Peptide Hydrolases
Bacteriophage T7
Bacteriophages
Inclusion Bodies
Ion Exchange Chromatography
Substrates
Substrate Specificity
Chromatography
Purification
Ion exchange
Plasmids
High Pressure Liquid Chromatography
Peptides

Keywords

  • Bovine leukemia virus
  • E. coli
  • Polyprotein precursor processing
  • Recombinant product accumulation
  • Retroviral proteinase
  • Synthetic peptide substrate

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Andreansky, M., Hrušková-Heidingsfeldová, O., Sedláčeka, J., Konvalinka, J., Bláha, I., Ječmen, P., ... Fábry, M. (1991). High-level expression of enzymatically active bovine leukemia virus proteinase in E. coli. FEBS Letters, 287(1-2), 129-132. https://doi.org/10.1016/0014-5793(91)80032-X

High-level expression of enzymatically active bovine leukemia virus proteinase in E. coli. / Andreansky, Martin; Hrušková-Heidingsfeldová, Olga; Sedláčeka, Juraj; Konvalinka, Jan; Bláha, Ivo; Ječmen, Petr; Hořejši, Magda; Štrop, Petr; Fábry, Milan.

In: FEBS Letters, Vol. 287, No. 1-2, 05.08.1991, p. 129-132.

Research output: Contribution to journalArticle

Andreansky, M, Hrušková-Heidingsfeldová, O, Sedláčeka, J, Konvalinka, J, Bláha, I, Ječmen, P, Hořejši, M, Štrop, P & Fábry, M 1991, 'High-level expression of enzymatically active bovine leukemia virus proteinase in E. coli', FEBS Letters, vol. 287, no. 1-2, pp. 129-132. https://doi.org/10.1016/0014-5793(91)80032-X
Andreansky M, Hrušková-Heidingsfeldová O, Sedláčeka J, Konvalinka J, Bláha I, Ječmen P et al. High-level expression of enzymatically active bovine leukemia virus proteinase in E. coli. FEBS Letters. 1991 Aug 5;287(1-2):129-132. https://doi.org/10.1016/0014-5793(91)80032-X
Andreansky, Martin ; Hrušková-Heidingsfeldová, Olga ; Sedláčeka, Juraj ; Konvalinka, Jan ; Bláha, Ivo ; Ječmen, Petr ; Hořejši, Magda ; Štrop, Petr ; Fábry, Milan. / High-level expression of enzymatically active bovine leukemia virus proteinase in E. coli. In: FEBS Letters. 1991 ; Vol. 287, No. 1-2. pp. 129-132.
@article{048a7b5e7055447ea12290a882f717eb,
title = "High-level expression of enzymatically active bovine leukemia virus proteinase in E. coli",
abstract = "An E. coli plasmid expressing efficiently an artificial precursor of bovine leukemia virus (BLV) proteinase under transcriptional control of the phage T7 promoter was constructed. The expression product accumulates in the induced E. coli cells in the form of insoluble cytoplasmic inclusions. Solubilization of the inclusions and a refolding step yield almost pure and completely self-processed proteinase. Purification to homogeneity was achieved by ion-exchange chromatography and reverse-phase HPLC. On a preparative scale, a high yield of enzymatically active proteinase was obtained. An initial study using a series of synthetic peptide substrates shows a distinct substrate specificity of BLV proteinase.",
keywords = "Bovine leukemia virus, E. coli, Polyprotein precursor processing, Recombinant product accumulation, Retroviral proteinase, Synthetic peptide substrate",
author = "Martin Andreansky and Olga Hruškov{\'a}-Heidingsfeldov{\'a} and Juraj Sedl{\'a}čeka and Jan Konvalinka and Ivo Bl{\'a}ha and Petr Ječmen and Magda Hořejši and Petr Štrop and Milan F{\'a}bry",
year = "1991",
month = "8",
day = "5",
doi = "10.1016/0014-5793(91)80032-X",
language = "English",
volume = "287",
pages = "129--132",
journal = "FEBS Letters",
issn = "0014-5793",
publisher = "Elsevier",
number = "1-2",

}

TY - JOUR

T1 - High-level expression of enzymatically active bovine leukemia virus proteinase in E. coli

AU - Andreansky, Martin

AU - Hrušková-Heidingsfeldová, Olga

AU - Sedláčeka, Juraj

AU - Konvalinka, Jan

AU - Bláha, Ivo

AU - Ječmen, Petr

AU - Hořejši, Magda

AU - Štrop, Petr

AU - Fábry, Milan

PY - 1991/8/5

Y1 - 1991/8/5

N2 - An E. coli plasmid expressing efficiently an artificial precursor of bovine leukemia virus (BLV) proteinase under transcriptional control of the phage T7 promoter was constructed. The expression product accumulates in the induced E. coli cells in the form of insoluble cytoplasmic inclusions. Solubilization of the inclusions and a refolding step yield almost pure and completely self-processed proteinase. Purification to homogeneity was achieved by ion-exchange chromatography and reverse-phase HPLC. On a preparative scale, a high yield of enzymatically active proteinase was obtained. An initial study using a series of synthetic peptide substrates shows a distinct substrate specificity of BLV proteinase.

AB - An E. coli plasmid expressing efficiently an artificial precursor of bovine leukemia virus (BLV) proteinase under transcriptional control of the phage T7 promoter was constructed. The expression product accumulates in the induced E. coli cells in the form of insoluble cytoplasmic inclusions. Solubilization of the inclusions and a refolding step yield almost pure and completely self-processed proteinase. Purification to homogeneity was achieved by ion-exchange chromatography and reverse-phase HPLC. On a preparative scale, a high yield of enzymatically active proteinase was obtained. An initial study using a series of synthetic peptide substrates shows a distinct substrate specificity of BLV proteinase.

KW - Bovine leukemia virus

KW - E. coli

KW - Polyprotein precursor processing

KW - Recombinant product accumulation

KW - Retroviral proteinase

KW - Synthetic peptide substrate

UR - http://www.scopus.com/inward/record.url?scp=0025886970&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0025886970&partnerID=8YFLogxK

U2 - 10.1016/0014-5793(91)80032-X

DO - 10.1016/0014-5793(91)80032-X

M3 - Article

VL - 287

SP - 129

EP - 132

JO - FEBS Letters

JF - FEBS Letters

SN - 0014-5793

IS - 1-2

ER -