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Recombinant expression of soluble murine prion protein for C‐terminal modification

Membrane attachment of prion protein (PrP) via its glycosylphosphatidylinositol (GPI) anchor plays a key role during conversion of cellular PrP into its pathogenic isoform PrP. Strategies to access homogenous lipidated PrP via expressed protein ligation (EPL) are required to fully decipher the effec... Full description

Journal Title: FEBS Letters 01 March 2013, Vol.587(5), pp.430-435
Main Author: Chu, Nam Ky
Other Authors: Becker, Christian F. W.
Format: Electronic Article Electronic Article
Language:
Subjects:
Asp
Cbd
Gpi
Gsh
Hdp
Nbd
Rml
Sbd
ID: ISSN: 0014-5793 ; E-ISSN: 1873-3468 ; DOI: 10.1016/j.febslet.2012.12.026
Zum Text:
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recordid: wj10.1016/j.febslet.2012.12.026
title: Recombinant expression of soluble murine prion protein for C‐terminal modification
format: Article
creator:
  • Chu, Nam Ky
  • Becker, Christian F. W.
subjects:
  • Asp
  • 6xhis-Atpase-Srta-Prp
  • Aspm
  • 6xhis-Atpase-Srta-Prp- Mxe Intein-6xhis-Cbd
  • Cbd
  • Chitin Binding Domain
  • Gpi
  • Glycosylphosphatidylinositol
  • Gsh
  • Reduced Glutathione
  • Gssg
  • Oxidized Glutathione
  • Hdp
  • 6xhis-Dnak-Tev-Prp
  • Iptg
  • Isopropyl-Β- D -Thiogalactopyranoside
  • Mesna
  • Sodium 2-Mercaptoethanesulfonate
  • Naoac
  • Sodium Acetate
  • Nbd
  • 4-Chloro-7-Nitrobenzofurazan
  • Pmsf
  • Phenylmethylsulfonyl Fluoride
  • Rml
  • Rocky Mountain Laboratories
  • Sbd
  • Substrate Binding Domain
  • Srta
  • Sortase A
  • Prion Protein
  • Soluble Expression
  • Semisynthesis
  • Disulfide Bridge
ispartof: FEBS Letters, 01 March 2013, Vol.587(5), pp.430-435
description: Membrane attachment of prion protein (PrP) via its glycosylphosphatidylinositol (GPI) anchor plays a key role during conversion of cellular PrP into its pathogenic isoform PrP. Strategies to access homogenous lipidated PrP via expressed protein ligation (EPL) are required to fully decipher the effect of membrane attachment. Such strategies suffer from insoluble expression of PrP‐intein fusion constructs and low folding efficiencies that severely limit the available amount of homogeneous lipidated PrP. Here, we describe an alternative method for expression of soluble PrP‐intein fusion proteins in that provides access to natively folded PrP ready to use in EPL. ► Generation of soluble PrP fusion proteins with DnaK and intein, respectively. ► Expression of up to 15 mg/l of soluble cellular prion proteins in . ► C‐terminal modification of cellular prion proteins without any refolding.
language:
source:
identifier: ISSN: 0014-5793 ; E-ISSN: 1873-3468 ; DOI: 10.1016/j.febslet.2012.12.026
fulltext: fulltext
issn:
  • 0014-5793
  • 00145793
  • 1873-3468
  • 18733468
url: Link


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titleRecombinant expression of soluble murine prion protein for C‐terminal modification
creatorChu, Nam Ky ; Becker, Christian F. W.
ispartofFEBS Letters, 01 March 2013, Vol.587(5), pp.430-435
identifier
subjectAsp ; 6xhis-Atpase-Srta-Prp ; Aspm ; 6xhis-Atpase-Srta-Prp- Mxe Intein-6xhis-Cbd ; Cbd ; Chitin Binding Domain ; Gpi ; Glycosylphosphatidylinositol ; Gsh ; Reduced Glutathione ; Gssg ; Oxidized Glutathione ; Hdp ; 6xhis-Dnak-Tev-Prp ; Iptg ; Isopropyl-Β- D -Thiogalactopyranoside ; Mesna ; Sodium 2-Mercaptoethanesulfonate ; Naoac ; Sodium Acetate ; Nbd ; 4-Chloro-7-Nitrobenzofurazan ; Pmsf ; Phenylmethylsulfonyl Fluoride ; Rml ; Rocky Mountain Laboratories ; Sbd ; Substrate Binding Domain ; Srta ; Sortase A ; Prion Protein ; Soluble Expression ; Semisynthesis ; Disulfide Bridge
descriptionMembrane attachment of prion protein (PrP) via its glycosylphosphatidylinositol (GPI) anchor plays a key role during conversion of cellular PrP into its pathogenic isoform PrP. Strategies to access homogenous lipidated PrP via expressed protein ligation (EPL) are required to fully decipher the effect of membrane attachment. Such strategies suffer from insoluble expression of PrP‐intein fusion constructs and low folding efficiencies that severely limit the available amount of homogeneous lipidated PrP. Here, we describe an alternative method for expression of soluble PrP‐intein fusion proteins in that provides access to natively folded PrP ready to use in EPL. ► Generation of soluble PrP fusion proteins with DnaK and intein, respectively. ► Expression of up to 15 mg/l of soluble cellular prion proteins in . ► C‐terminal modification of cellular prion proteins without any refolding.
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titleRecombinant expression of soluble murine prion protein for C‐terminal modification
descriptionMembrane attachment of prion protein (PrP) via its glycosylphosphatidylinositol (GPI) anchor plays a key role during conversion of cellular PrP into its pathogenic isoform PrP. Strategies to access homogenous lipidated PrP via expressed protein ligation (EPL) are required to fully decipher the effect of membrane attachment. Such strategies suffer from insoluble expression of PrP‐intein fusion constructs and low folding efficiencies that severely limit the available amount of homogeneous lipidated PrP. Here, we describe an alternative method for expression of soluble PrP‐intein fusion proteins in that provides access to natively folded PrP ready to use in EPL. ► Generation of soluble PrP fusion proteins with DnaK and intein, respectively. ► Expression of up to 15 mg/l of soluble cellular prion proteins in . ► C‐terminal modification of cellular prion proteins without any refolding.
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16xhis-Atpase-Srta-Prp
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36xhis-Atpase-Srta-Prp- Mxe Intein-6xhis-Cbd
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136xhis-Dnak-Tev-Prp
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214-Chloro-7-Nitrobenzofurazan
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32Semisynthesis
33Disulfide Bridge
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titleRecombinant expression of soluble murine prion protein for C‐terminal modification
authorChu, Nam Ky ; Becker, Christian F. W.
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214-Chloro-7-Nitrobenzofurazan
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abstractMembrane attachment of prion protein (PrP) via its glycosylphosphatidylinositol (GPI) anchor plays a key role during conversion of cellular PrP into its pathogenic isoform PrP. Strategies to access homogenous lipidated PrP via expressed protein ligation (EPL) are required to fully decipher the effect of membrane attachment. Such strategies suffer from insoluble expression of PrP‐intein fusion constructs and low folding efficiencies that severely limit the available amount of homogeneous lipidated PrP. Here, we describe an alternative method for expression of soluble PrP‐intein fusion proteins in that provides access to natively folded PrP ready to use in EPL. ► Generation of soluble PrP fusion proteins with DnaK and intein, respectively. ► Expression of up to 15 mg/l of soluble cellular prion proteins in . ► C‐terminal modification of cellular prion proteins without any refolding.
doi10.1016/j.febslet.2012.12.026
pages430-435
date2013-03-01