Expression of the murine leukemia virus protease in fusion with maltose-binding protein in Escherichia coli

Anita Fehér, Péter Boross, Tamás Sperka, Stephen Oroszlan, József Tözsér

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

The protease of murine leukemia virus (MLV) was cloned into pMal-c2 vector, expressed in fusion with maltose-binding protein (MBP), and purified to homogeneity after Factor Xa cleavage of the chimeric protein. Substantial degradation of the fusion protein was observed during expression, which severely diminished the yield. The degree of degradation of the fusion protein was even more pronounced when a single-chain form of the MLV protease was cloned after the gene coding for MBP. To increase the yield, a hexahistidine tag with an additional Factor Xa cleavage site was cloned after the protease and nickel chelate affinity chromatography was used as the first purification step. The modified procedure resulted in substantially higher yield as compared to the original procedure. The degradation of hexahistidine-tagged active site mutant MLV protease was very low and comparable to that obtained with hexahistidine-tagged MBP, but purified MLV protease alone was not able to degrade purified MBP, suggesting that during expression the active MLV protease may activate bacterial proteases which appear to be responsible for the degradation of the fusion proteins.

Original languageEnglish
Pages (from-to)62-68
Number of pages7
JournalProtein Expression and Purification
Volume35
Issue number1
DOIs
Publication statusPublished - May 2004

Fingerprint

Maltose-Binding Proteins
Murine Leukemia Viruses
His-His-His-His-His-His
Viruses
Escherichia coli
Peptide Hydrolases
Fusion reactions
Proteolysis
Degradation
Factor Xa
Proteins
Affinity chromatography
Nickel
Affinity Chromatography
Purification
Catalytic Domain
Genes

Keywords

  • MLV
  • Purification
  • Retroviral protease

ASJC Scopus subject areas

  • Biochemistry

Cite this

Expression of the murine leukemia virus protease in fusion with maltose-binding protein in Escherichia coli. / Fehér, Anita; Boross, Péter; Sperka, Tamás; Oroszlan, Stephen; Tözsér, József.

In: Protein Expression and Purification, Vol. 35, No. 1, 05.2004, p. 62-68.

Research output: Contribution to journalArticle

Fehér, Anita ; Boross, Péter ; Sperka, Tamás ; Oroszlan, Stephen ; Tözsér, József. / Expression of the murine leukemia virus protease in fusion with maltose-binding protein in Escherichia coli. In: Protein Expression and Purification. 2004 ; Vol. 35, No. 1. pp. 62-68.
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