Preparation of bioactive soluble human leukemia inhibitory factor from recombinant Escherichia coli using thioredoxin as fusion partner.

2.50
Hdl Handle:
http://hdl.handle.net/10033/135509
Title:
Preparation of bioactive soluble human leukemia inhibitory factor from recombinant Escherichia coli using thioredoxin as fusion partner.
Authors:
Tomala, Magda; Lavrentieva, Antonina; Moretti, Pierre; Rinas, Ursula; Kasper, Cornelia; Stahl, Frank; Schambach, Axel; Warlich, Eva; Martin, Ulrich; Cantz, Tobias; Scheper, Thomas
Abstract:
Leukemia inhibitory factor (LIF) is a polyfunctional cytokine with numerous regulatory effects in vivo and in vitro. In stem cell cultures it is the essential media supplement for the maintenance of pluripotency of embryonic and induced pluripotent stem cells. With regard to large scale cultures of these cells, LIF is needed in high quality and quantity and represents the major cost determining factor (90%) of the culture media. In this report, we describe a novel production and purification process for human LIF (hLIF) from recombinant Escherichia coli cultures. hLIF was cloned into pET32b and expressed as soluble protein in fusion with thioredoxin. After purification based on membrane adsorber technology, the fusion protein was cleaved using TEV protease. Released, soluble hLIF was subsequently purified by cation exchange chromatography and successfully tested for its biological activity using suspension cultures of murine embryonic and induced pluripotent stem cells. Our novel protocol for the production of recombinant hLIF is very suitable and effective for the production of poorly soluble proteins through expression in fusion with the solubilizing partner thioredoxin.
Affiliation:
Institute of Technical Chemistry, Leibniz University of Hannover, Callinstr. 5, 30167 Hannover, Germany.
Citation:
Preparation of bioactive soluble human leukemia inhibitory factor from recombinant Escherichia coli using thioredoxin as fusion partner. 2010, 73 (1):51-7 Protein Expr. Purif.
Journal:
Protein expression and purification
Issue Date:
Sep-2010
URI:
http://hdl.handle.net/10033/135509
DOI:
10.1016/j.pep.2010.04.002
PubMed ID:
20381622
Type:
Article
Language:
en
ISSN:
1096-0279
Appears in Collections:
publications of the research group recombinant protein expression (RPEX)

Full metadata record

DC FieldValue Language
dc.contributor.authorTomala, Magdaen
dc.contributor.authorLavrentieva, Antoninaen
dc.contributor.authorMoretti, Pierreen
dc.contributor.authorRinas, Ursulaen
dc.contributor.authorKasper, Corneliaen
dc.contributor.authorStahl, Franken
dc.contributor.authorSchambach, Axelen
dc.contributor.authorWarlich, Evaen
dc.contributor.authorMartin, Ulrichen
dc.contributor.authorCantz, Tobiasen
dc.contributor.authorScheper, Thomasen
dc.date.accessioned2011-07-07T08:11:30Z-
dc.date.available2011-07-07T08:11:30Z-
dc.date.issued2010-09-
dc.identifier.citationPreparation of bioactive soluble human leukemia inhibitory factor from recombinant Escherichia coli using thioredoxin as fusion partner. 2010, 73 (1):51-7 Protein Expr. Purif.en
dc.identifier.issn1096-0279-
dc.identifier.pmid20381622-
dc.identifier.doi10.1016/j.pep.2010.04.002-
dc.identifier.urihttp://hdl.handle.net/10033/135509-
dc.description.abstractLeukemia inhibitory factor (LIF) is a polyfunctional cytokine with numerous regulatory effects in vivo and in vitro. In stem cell cultures it is the essential media supplement for the maintenance of pluripotency of embryonic and induced pluripotent stem cells. With regard to large scale cultures of these cells, LIF is needed in high quality and quantity and represents the major cost determining factor (90%) of the culture media. In this report, we describe a novel production and purification process for human LIF (hLIF) from recombinant Escherichia coli cultures. hLIF was cloned into pET32b and expressed as soluble protein in fusion with thioredoxin. After purification based on membrane adsorber technology, the fusion protein was cleaved using TEV protease. Released, soluble hLIF was subsequently purified by cation exchange chromatography and successfully tested for its biological activity using suspension cultures of murine embryonic and induced pluripotent stem cells. Our novel protocol for the production of recombinant hLIF is very suitable and effective for the production of poorly soluble proteins through expression in fusion with the solubilizing partner thioredoxin.en
dc.language.isoenen
dc.subject.meshAnimalsen
dc.subject.meshAntigens, CD15en
dc.subject.meshBase Sequenceen
dc.subject.meshCell Lineen
dc.subject.meshCell Proliferationen
dc.subject.meshCloning, Molecularen
dc.subject.meshElectrophoresis, Polyacrylamide Gelen
dc.subject.meshEmbryonic Stem Cellsen
dc.subject.meshEndopeptidasesen
dc.subject.meshEscherichia colien
dc.subject.meshFlow Cytometryen
dc.subject.meshHumansen
dc.subject.meshLeukemia Inhibitory Factoren
dc.subject.meshMiceen
dc.subject.meshMolecular Sequence Dataen
dc.subject.meshRecombinant Fusion Proteinsen
dc.subject.meshSolubilityen
dc.subject.meshThioredoxinsen
dc.titlePreparation of bioactive soluble human leukemia inhibitory factor from recombinant Escherichia coli using thioredoxin as fusion partner.en
dc.typeArticleen
dc.contributor.departmentInstitute of Technical Chemistry, Leibniz University of Hannover, Callinstr. 5, 30167 Hannover, Germany.en
dc.identifier.journalProtein expression and purificationen
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