Improved purification protocol for wild-type and mutant human foamy virus proteases

Péter Boross, J. Tőzsér, P. Bagossi

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

Wild-type and an active site mutant (S25T) human foamy virus (HFV) proteases were expressed in fusion with maltose binding protein in Escherichia coli. The mutant enzyme contained a Ser to Thr mutation in the -Asp-Ser-Gly- active site triplet of the enzyme, which forms the "fireman's grip" between the two subunits of the homodimeric enzyme. The fusion proteins were purified by affinity chromatography on amylose resin, cleaved with factor Xa, and the processed enzymes were purified by gel filtration under denaturing condition. Refolding after purification resulted in active enzymes with comparable yields. Furthermore, both enzymes showed similar catalytic activities in an oligopeptide substrate representing an HFV Gag cleavage site. However, the S25T mutant showed increased stability in urea unfolding experiment, in a good agreement with the suggested role of the Thr residue of fireman's grip.

Original languageEnglish
Pages (from-to)343-347
Number of pages5
JournalProtein Expression and Purification
Volume46
Issue number2
DOIs
Publication statusPublished - Apr 2006

Fingerprint

Simian foamy virus
Viruses
Purification
Peptide Hydrolases
Enzymes
Hand Strength
Catalytic Domain
Fusion reactions
Maltose-Binding Proteins
Affinity chromatography
Oligopeptides
Amylose
Factor Xa
Affinity Chromatography
Escherichia coli
Gel Chromatography
Urea
Catalyst activity
Resins
Gels

Keywords

  • Enzyme kinetics
  • Human foamy virus protease
  • Mutagenesis
  • Urea unfolding

ASJC Scopus subject areas

  • Biochemistry

Cite this

Improved purification protocol for wild-type and mutant human foamy virus proteases. / Boross, Péter; Tőzsér, J.; Bagossi, P.

In: Protein Expression and Purification, Vol. 46, No. 2, 04.2006, p. 343-347.

Research output: Contribution to journalArticle

@article{2077e224f2f64b498cdb916a2faada07,
title = "Improved purification protocol for wild-type and mutant human foamy virus proteases",
abstract = "Wild-type and an active site mutant (S25T) human foamy virus (HFV) proteases were expressed in fusion with maltose binding protein in Escherichia coli. The mutant enzyme contained a Ser to Thr mutation in the -Asp-Ser-Gly- active site triplet of the enzyme, which forms the {"}fireman's grip{"} between the two subunits of the homodimeric enzyme. The fusion proteins were purified by affinity chromatography on amylose resin, cleaved with factor Xa, and the processed enzymes were purified by gel filtration under denaturing condition. Refolding after purification resulted in active enzymes with comparable yields. Furthermore, both enzymes showed similar catalytic activities in an oligopeptide substrate representing an HFV Gag cleavage site. However, the S25T mutant showed increased stability in urea unfolding experiment, in a good agreement with the suggested role of the Thr residue of fireman's grip.",
keywords = "Enzyme kinetics, Human foamy virus protease, Mutagenesis, Urea unfolding",
author = "P{\'e}ter Boross and J. Tőzs{\'e}r and P. Bagossi",
year = "2006",
month = "4",
doi = "10.1016/j.pep.2005.09.004",
language = "English",
volume = "46",
pages = "343--347",
journal = "Protein Expression and Purification",
issn = "1046-5928",
publisher = "Academic Press Inc.",
number = "2",

}

TY - JOUR

T1 - Improved purification protocol for wild-type and mutant human foamy virus proteases

AU - Boross, Péter

AU - Tőzsér, J.

AU - Bagossi, P.

PY - 2006/4

Y1 - 2006/4

N2 - Wild-type and an active site mutant (S25T) human foamy virus (HFV) proteases were expressed in fusion with maltose binding protein in Escherichia coli. The mutant enzyme contained a Ser to Thr mutation in the -Asp-Ser-Gly- active site triplet of the enzyme, which forms the "fireman's grip" between the two subunits of the homodimeric enzyme. The fusion proteins were purified by affinity chromatography on amylose resin, cleaved with factor Xa, and the processed enzymes were purified by gel filtration under denaturing condition. Refolding after purification resulted in active enzymes with comparable yields. Furthermore, both enzymes showed similar catalytic activities in an oligopeptide substrate representing an HFV Gag cleavage site. However, the S25T mutant showed increased stability in urea unfolding experiment, in a good agreement with the suggested role of the Thr residue of fireman's grip.

AB - Wild-type and an active site mutant (S25T) human foamy virus (HFV) proteases were expressed in fusion with maltose binding protein in Escherichia coli. The mutant enzyme contained a Ser to Thr mutation in the -Asp-Ser-Gly- active site triplet of the enzyme, which forms the "fireman's grip" between the two subunits of the homodimeric enzyme. The fusion proteins were purified by affinity chromatography on amylose resin, cleaved with factor Xa, and the processed enzymes were purified by gel filtration under denaturing condition. Refolding after purification resulted in active enzymes with comparable yields. Furthermore, both enzymes showed similar catalytic activities in an oligopeptide substrate representing an HFV Gag cleavage site. However, the S25T mutant showed increased stability in urea unfolding experiment, in a good agreement with the suggested role of the Thr residue of fireman's grip.

KW - Enzyme kinetics

KW - Human foamy virus protease

KW - Mutagenesis

KW - Urea unfolding

UR - http://www.scopus.com/inward/record.url?scp=33645406938&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33645406938&partnerID=8YFLogxK

U2 - 10.1016/j.pep.2005.09.004

DO - 10.1016/j.pep.2005.09.004

M3 - Article

C2 - 16243539

AN - SCOPUS:33645406938

VL - 46

SP - 343

EP - 347

JO - Protein Expression and Purification

JF - Protein Expression and Purification

SN - 1046-5928

IS - 2

ER -