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Efficient production of bioactive recombinant human Flt3 ligand in E. coli

Kenneth Verstraete (UGent) , Sina Koch, Sevgi Ertugrul, Isabel Vandenberghe (UGent) , Maarten Aerts (UGent) , Gonzalez Vandriessche, Christian Thiede and Savvas Savvides (UGent)
(2009) PROTEIN JOURNAL. 28(2). p.57-65
Author
Organization
Abstract
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.
Keywords
In vitro refolding, Protein purification, Recombinant, Flt3 ligand, 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

Citation

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MLA
Verstraete, Kenneth et al. “Efficient Production of Bioactive Recombinant Human Flt3 Ligand in E. Coli.” PROTEIN JOURNAL 28.2 (2009): 57–65. Print.
APA
Verstraete, K., Koch, S., Ertugrul, S., Vandenberghe, I., Aerts, M., Vandriessche, G., Thiede, C., et al. (2009). Efficient production of bioactive recombinant human Flt3 ligand in E. coli. PROTEIN JOURNAL, 28(2), 57–65.
Chicago author-date
Verstraete, Kenneth, Sina Koch, Sevgi Ertugrul, Isabel Vandenberghe, Maarten Aerts, Gonzalez Vandriessche, Christian Thiede, and Savvas Savvides. 2009. “Efficient Production of Bioactive Recombinant Human Flt3 Ligand in E. Coli.” Protein Journal 28 (2): 57–65.
Chicago author-date (all authors)
Verstraete, Kenneth, Sina Koch, Sevgi Ertugrul, Isabel Vandenberghe, Maarten Aerts, Gonzalez Vandriessche, Christian Thiede, and Savvas Savvides. 2009. “Efficient Production of Bioactive Recombinant Human Flt3 Ligand in E. Coli.” Protein Journal 28 (2): 57–65.
Vancouver
1.
Verstraete K, Koch S, Ertugrul S, Vandenberghe I, Aerts M, Vandriessche G, et al. Efficient production of bioactive recombinant human Flt3 ligand in E. coli. PROTEIN JOURNAL. 2009;28(2):57–65.
IEEE
[1]
K. Verstraete et al., “Efficient production of bioactive recombinant human Flt3 ligand in E. coli,” PROTEIN JOURNAL, vol. 28, no. 2, pp. 57–65, 2009.
@article{8071298,
  abstract     = {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.},
  author       = {Verstraete, Kenneth and Koch, Sina and Ertugrul, Sevgi and Vandenberghe, Isabel and Aerts, Maarten and Vandriessche, Gonzalez and Thiede, Christian and Savvides, Savvas},
  issn         = {1572-3887},
  journal      = {PROTEIN JOURNAL},
  keywords     = {In vitro refolding,Protein purification,Recombinant,Flt3 ligand,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},
  language     = {eng},
  number       = {2},
  pages        = {57--65},
  title        = {Efficient production of bioactive recombinant human Flt3 ligand in E. coli},
  url          = {http://dx.doi.org/10.1007/s10930-009-9164-5},
  volume       = {28},
  year         = {2009},
}
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