Expression and purification of bioactive soluble murine stem cell factor from recombinant Escherichia coli using thioredoxin as fusion partner.

2.50
Hdl Handle:
http://hdl.handle.net/10033/135105
Title:
Expression and purification of bioactive soluble murine stem cell factor from recombinant Escherichia coli using thioredoxin as fusion partner.
Authors:
Bals, Carola; Schambach, Axel; Meyer, Johann; Scheper, Thomas; Rinas, Ursula
Abstract:
Stem cell factor (SCF) known as the c-kit ligand, plays important roles in spermatogenesis, melanogenesis and early stages of hematopoiesis. As for the latter, SCF is essential for growth and expansion of hematopoietic stem and progenitor cells. We herein describe the production of recombinant murine SCF from Escherichia coli as soluble thioredoxin-fusion protein. The formation of insoluble and inactive inclusion bodies, usually observed when SCF is expressed in E. coli, was almost entirely prevented. After purification based on membrane adsorber technology, the fusion protein was subsequently cleaved by TEV protease in order to release mature mSCF. Following dialysis and a final purification step, the target protein was isolated in high purity. Bioactivity of mSCF was proven by different tests (MTT analogous assay, long-term proliferation assay) applying a human megakaryocytic cell line. Furthermore, the biological activity of the uncleaved fusion protein was tested as well. We observed a significant activity, even though it was less than the activity displayed by the purified mSCF. In summary, avoiding inclusion body formation we present an efficient production procedure for mSCF, one of the most important stem cell cytokines.
Affiliation:
Excellence Cluster Rebirth, Institute of Technical Chemistry-Life Science, Leibniz University of Hannover, Callinstr.5, 30167 Hannover, Germany.
Citation:
Expression and purification of bioactive soluble murine stem cell factor from recombinant Escherichia coli using thioredoxin as fusion partner. 2011, 152 (1-2):1-8 J. Biotechnol.
Journal:
Journal of biotechnology
Issue Date:
10-Mar-2011
URI:
http://hdl.handle.net/10033/135105
DOI:
10.1016/j.jbiotec.2011.01.012
PubMed ID:
21262286
Type:
Article
Language:
en
ISSN:
1873-4863
Appears in Collections:
Publications of the RG Rekombinante Proteinexpression (RPEX)

Full metadata record

DC FieldValue Language
dc.contributor.authorBals, Carolaen
dc.contributor.authorSchambach, Axelen
dc.contributor.authorMeyer, Johannen
dc.contributor.authorScheper, Thomasen
dc.contributor.authorRinas, Ursulaen
dc.date.accessioned2011-07-01T13:45:04Z-
dc.date.available2011-07-01T13:45:04Z-
dc.date.issued2011-03-10-
dc.identifier.citationExpression and purification of bioactive soluble murine stem cell factor from recombinant Escherichia coli using thioredoxin as fusion partner. 2011, 152 (1-2):1-8 J. Biotechnol.en
dc.identifier.issn1873-4863-
dc.identifier.pmid21262286-
dc.identifier.doi10.1016/j.jbiotec.2011.01.012-
dc.identifier.urihttp://hdl.handle.net/10033/135105-
dc.description.abstractStem cell factor (SCF) known as the c-kit ligand, plays important roles in spermatogenesis, melanogenesis and early stages of hematopoiesis. As for the latter, SCF is essential for growth and expansion of hematopoietic stem and progenitor cells. We herein describe the production of recombinant murine SCF from Escherichia coli as soluble thioredoxin-fusion protein. The formation of insoluble and inactive inclusion bodies, usually observed when SCF is expressed in E. coli, was almost entirely prevented. After purification based on membrane adsorber technology, the fusion protein was subsequently cleaved by TEV protease in order to release mature mSCF. Following dialysis and a final purification step, the target protein was isolated in high purity. Bioactivity of mSCF was proven by different tests (MTT analogous assay, long-term proliferation assay) applying a human megakaryocytic cell line. Furthermore, the biological activity of the uncleaved fusion protein was tested as well. We observed a significant activity, even though it was less than the activity displayed by the purified mSCF. In summary, avoiding inclusion body formation we present an efficient production procedure for mSCF, one of the most important stem cell cytokines.en
dc.language.isoenen
dc.subject.meshAnimalsen
dc.subject.meshCell Lineen
dc.subject.meshCell Proliferationen
dc.subject.meshElectrophoresis, Polyacrylamide Gelen
dc.subject.meshEscherichia colien
dc.subject.meshGenetic Vectorsen
dc.subject.meshHumansen
dc.subject.meshMiceen
dc.subject.meshPolymerase Chain Reactionen
dc.subject.meshStem Cell Factoren
dc.subject.meshThioredoxinsen
dc.titleExpression and purification of bioactive soluble murine stem cell factor from recombinant Escherichia coli using thioredoxin as fusion partner.en
dc.typeArticleen
dc.contributor.departmentExcellence Cluster Rebirth, Institute of Technical Chemistry-Life Science, Leibniz University of Hannover, Callinstr.5, 30167 Hannover, Germany.en
dc.identifier.journalJournal of biotechnologyen

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