Loading...
Expression and purification of bioactive soluble murine stem cell factor from recombinant Escherichia coli using thioredoxin as fusion partner.
Bals, Carola Schambach, Axel Meyer, Johann Scheper, Thomas Rinas, Ursula
Bals, Carola
Schambach, Axel
Meyer, Johann
Scheper, Thomas
Rinas, Ursula
Citations
Altmetric:
Advisors
Editors
Other Contributors
Issue Date
2011-03-10
Submitted date
Files
Loading...
original manuscript
Adobe PDF, 703.52 KB
Other Titles
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.
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.
Publisher
Journal
PubMed ID
PubMed Central ID
Additional Links
Embedded video
Type
Article
Language
en
Description
Series/Report no.
ISSN
1873-4863