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primary topic: http://crdd.osdd.net/open/id/eprint/187

http://crdd.osdd.net/open/id/eprint/187
type: AcademicArticle
type: Article
title: Direct proteolysis-based purification of an overexpressed hyperthermophile protein from Escherichia coli lysate: a novel exploitation of the link between structural stability and proteolytic resistance.
abstract: The susceptibility of a peptide bond to cleavage by a protease is determined by: (a) the flexibility of the protein chain region in which it is located, (b) the extent to which the bond is exposed, and (c) the nature of the local interactions made by the sidechains of its flanking residues. Each of these parameters is known to be influenced by the overall structural stability of the protein; thus, proteins of higher structurally stability commonly show higher resistance to proteolysis. Extrapolating this relationship to 'ultrastable' proteins, our intention here was to investigate whether a hyperthermophile protein expressed and folded within Escherichia coli could prove to be so resistant to proteolysis as to allow direct purification from complex mixtures of E. coli cytoplasmic and/or membrane proteins, through proteolytic means. Thus, we cloned the gene encoding the triosephosphate isomerase enzyme of Pyrococcus furiosus (PfuTIM) and overexpressed it in E. coli in fusion with glutathione S-transferase (GST). The GST-PfuTIM fusion product partitioned mainly into the insoluble fraction of the whole cell lysate. Upon exposure of the E. coli cell lysate precipitate fractions to the non-specific protease, subtilisin, all polypeptides barring PfuTIM (including the GST affinity tag cloned in fusion with PfuTIM) were found to be degraded to undetectable levels. Trace residual amounts of an E. coli protein, OmpF, survived proteolytic digestion, together with an extremely pure population of PfuTIM. Either autonomously or in combination with the more conventional method of heating solutions to enrich heat-stable proteins through the thermal unfolding and aggregation of all other proteins, such proteolysis-based purification could prove to be useful.
date: 2005-03
volume: 40
issue: 1
is part of: Protein expression and purification
status: peerReviewed
status: published
author list: authors
type: EPrint
type: ArticleEPrint
is part of: repository
has document: http://crdd.osdd.net/open/id/document/2180
has version: http://crdd.osdd.net/open/id/document/2180
has published: http://crdd.osdd.net/open/id/document/2180
has document: http://crdd.osdd.net/open/id/document/2187
has document: http://crdd.osdd.net/open/id/document/2188
has document: http://crdd.osdd.net/open/id/document/2189
has document: http://crdd.osdd.net/open/id/document/2190
has document: http://crdd.osdd.net/open/id/document/2191
subject: QR Microbiology

http://crdd.osdd.net/open/id/document/2180
type: Document
label: Direct proteolysis-based purification of an overexpressed hyperthermophile protein from Escherichia coli lysate: a novel exploitation of the link between structural stability and proteolytic resistance. (PDF)

Elsevier Science
type: Organization

Protein expression and purification
type: Collection
issn: 10465928

Sourav Mukherjee
type: Person
given name: Sourav
family name: Mukherjee

Purnananda Guptasarma
type: Person
given name: Purnananda
family name: Guptasarma

http://crdd.osdd.net/open/id/document/2187
type: Document
label: Direct proteolysis-based purification of an overexpressed hyperthermophile protein from Escherichia coli lysate: a novel exploitation of the link between structural stability and proteolytic resistance. (Image (PNG))
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http://crdd.osdd.net/open/id/document/2188
type: Document
label: Direct proteolysis-based purification of an overexpressed hyperthermophile protein from Escherichia coli lysate: a novel exploitation of the link between structural stability and proteolytic resistance. (Image (PNG))
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http://crdd.osdd.net/open/id/document/2189
type: Document
label: Direct proteolysis-based purification of an overexpressed hyperthermophile protein from Escherichia coli lysate: a novel exploitation of the link between structural stability and proteolytic resistance. (Image (PNG))
is version of: http://crdd.osdd.net/open/id/document/2180
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http://crdd.osdd.net/open/id/document/2190
type: Document
label: Direct proteolysis-based purification of an overexpressed hyperthermophile protein from Escherichia coli lysate: a novel exploitation of the link between structural stability and proteolytic resistance. (Image (PNG))
is version of: http://crdd.osdd.net/open/id/document/2180
is volatile version of: http://crdd.osdd.net/open/id/document/2180
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http://crdd.osdd.net/open/id/document/2191
type: Document
label: Direct proteolysis-based purification of an overexpressed hyperthermophile protein from Escherichia coli lysate: a novel exploitation of the link between structural stability and proteolytic resistance. (Indexer Terms)
is version of: http://crdd.osdd.net/open/id/document/2180
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http://crdd.osdd.net/open/187/
title: HTML Summary of #187 Direct proteolysis-based purification of an overexpressed hyperthermophile protein from Escherichia coli lysate: a novel exploitation of the link between structural stability and proteolytic resistance.
format: text/html
primary topic: http://crdd.osdd.net/open/id/eprint/187

QR Microbiology
type: Concept