Expression and purification of bioactive soluble murine stem cell factor from recombinant Escherichia coli using thioredoxin as fusion partner.
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.
Excellence Cluster Rebirth, Institute of Technical Chemistry-Life Science, Leibniz University of Hannover, Callinstr.5, 30167 Hannover, Germany.
Carola
C
Bals
Axel
A
Schambach
Johann
J
Meyer
Thomas
T
Scheper
Ursula
U
Rinas
D000818:Animals; D002460:Cell Line; D049109:Cell Proliferation; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D051379:Mice; D016133:Polymerase Chain Reaction; D019089:Stem Cell Factor / Q000235:genetics / Q000378:metabolism*; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism*
D019089:Stem Cell Factor; D013879:Thioredoxins