Efficient Production of Bioactive Recombinant Human Flt3 Ligand in E. coli

Verstraete K., Koch S., Ertugrul S., Vandenberghe I., Aerts M., Vandriessche G., ...Daha Fazla

PROTEIN JOURNAL, cilt.28, sa.2, ss.57-65, 2009 (SCI-Expanded) identifier identifier identifier

  • Yayın Türü: Makale / Tam Makale
  • Cilt numarası: 28 Sayı: 2
  • Basım Tarihi: 2009
  • Doi Numarası: 10.1007/s10930-009-9164-5
  • Dergi Adı: PROTEIN JOURNAL
  • Derginin Tarandığı İndeksler: Science Citation Index Expanded (SCI-EXPANDED), Scopus
  • Sayfa Sayıları: ss.57-65
  • Anahtar Kelimeler: Flt3 ligand, Recombinant, In vitro refolding, Protein purification, Bioactivity assays, RECEPTOR TYROSINE KINASE, MYELOID-LEUKEMIA CELLS, COLONY-STIMULATING FACTOR, HEMATOPOIETIC STEM-CELLS, BONE-MARROW, FLT3/FLK2 LIGAND, DENDRITIC CELLS, IN-VIVO, PROTEINS, PROLIFERATION
  • Ankara Üniversitesi Adresli: Evet

Özet

Flt3 ligand (FL) is an early-acting hematopoietic cytokine that stimulates the proliferation and differentiation of hematopoietic progenitor cells by activating its cognate receptor, Flt3. Recently, FL was shown to potently contribute to the development and expansion of anti gen-presenting dendritic cells and CD34(+) natural killer cell progenitors in vivo. Here, we report a comprehensive method for the production of bioactive recombinant human FL (rhFL) in E. coli, suitable for structural, biophysical and physiological studies. A soluble form of human FL capable of binding to the Ftl3 receptor could be overexpressed in the E. coli strain Rosetta-gami(DE3) as inclusion bodies. We have established protocols for the efficient in vitro refolding and ensuing purification of rhFL to homogeneity (>95%), with yields approaching 5 mg Of pure rhFL per liter of culture. The ability of rhFL to adopt a bioactive conformation was confirmed via a cell-proliferation assay and the activation of the Flt3 receptor in the human leukemic cell line, OCI-AML3.