Details
| Originalsprache | Englisch |
|---|---|
| Seiten (von - bis) | 1-8 |
| Seitenumfang | 8 |
| Fachzeitschrift | Journal of biotechnology |
| Jahrgang | 152 |
| Ausgabenummer | 1-2 |
| Publikationsstatus | Veröffentlicht - 22 Jan. 2011 |
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.
ASJC Scopus Sachgebiete
- Biochemie, Genetik und Molekularbiologie (insg.)
- Biotechnologie
- Chemische Verfahrenstechnik (insg.)
- Bioengineering
- Immunologie und Mikrobiologie (insg.)
- Angewandte Mikrobiologie und Biotechnologie
Zitieren
- Standard
- Harvard
- Apa
- Vancouver
- BibTex
- RIS
in: Journal of biotechnology, Jahrgang 152, Nr. 1-2, 22.01.2011, S. 1-8.
Publikation: Beitrag in Fachzeitschrift › Artikel › Forschung › Peer-Review
}
TY - JOUR
T1 - Expression and purification of bioactive soluble murine stem cell factor from recombinant Escherichia coli using thioredoxin as fusion partner
AU - Bals, Carola
AU - Schambach, Axel
AU - Meyer, Johann
AU - Scheper, Thomas
AU - Rinas, Ursula
N1 - Funding information: This work was partially supported by funds from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) for the Cluster of Excellence RERBIRTH (Exc 62/1).
PY - 2011/1/22
Y1 - 2011/1/22
N2 - 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.
AB - 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.
KW - Escherichia coli
KW - Fusion protein
KW - Membrane adsorber technology
KW - Soluble protein expression
KW - Stem cell factor
KW - Thioredoxin
UR - http://www.scopus.com/inward/record.url?scp=79952042906&partnerID=8YFLogxK
U2 - 10.1016/j.jbiotec.2011.01.012
DO - 10.1016/j.jbiotec.2011.01.012
M3 - Article
C2 - 21262286
AN - SCOPUS:79952042906
VL - 152
SP - 1
EP - 8
JO - Journal of biotechnology
JF - Journal of biotechnology
SN - 0168-1656
IS - 1-2
ER -