EXPRESSION AND PURIFICATION OF BIOLOGICALLY ACTIVE RECOMBINANT QUAIL STEM CELL FACTOR IN E. COLI

✍ Scribed by Susan D'Costa; Michael J. Kulik; James N. Petitte

• Publisher: Elsevier Science
• Year: 2000
• Tongue: English
• Weight: 775 KB
• Volume: 24
• Category: Article
• ISSN: 1065-6995
• DOI: 10.1006/cbir.1999.0500

✦ Synopsis

Abstract

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 22–23kD 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 50ng/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.