Expression and purification of biologically active recombinant quail stem cell factor in E. coli.
Stem cell factor (SCF) is a multifunctional cytokine involved in hematopoiesis, melanogenesis and gametogenesis. Previous studies have demonstrated that avian SCF is a requirement for the proliferation and survival of various cell types in vivo and in vitro. In the current study, recombinant quail stem cell factor was produced in Escherichia coli using a prokaryotic expression system. SCF was expressed as a fusion protein with a histidine hexamer tag at the N-terminal end of the protein. Following expression, the protein was purified by affinity chromatography on the Ni-NTA column. The uninduced and induced protein lysates and the purified protein were separated by SDS-PAGE and transferred onto nitrocellulose membrane. Western blot analysis with the monoclonal antibody to the histidine tag identified SCF in the induced cell lysates and the purified sample. The recombinant SCF was approximately 22-23 kD in size. This protein was generated devoid of the signal peptide, the transmembrane domain, and the intracellular domain and, hence, resembles the soluble form of SCF. Biological activity was assayed using the in vitro survival of E12 chicken dorsal root ganglion-derived sensory neurons. The addition of recombinant quail SCF improved neuronal survival. Survival (20.6%) was the highest at the 50 ng/ml concentration of SCF. The availability of quail SCF will be a valuable tool to further resolve the function of stem cell factor in birds.
D'Costa
S
S
Department of Poultry Science, North Carolina State University, Raleigh, NC 27695-7608, USA.
Kulik
M J
MJ
Petitte
J N
JN
D000818:Animals; D001681:Biological Assay; D015153:Blotting, Western; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002642:Chick Embryo; D002846:Chromatography, Affinity; D020934:Ciliary Neurotrophic Factor / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005727:Ganglia, Spinal / Q000166:cytology / Q000187:drug effects; D009474:Neurons / Q000166:cytology / Q000187:drug effects; D011784:Quail / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D019089:Stem Cell Factor / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology
D020934:Ciliary Neurotrophic Factor; D011993:Recombinant Fusion Proteins; D019089:Stem Cell Factor