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

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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 antigen-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.

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Abbreviations

Flt3:

FMS-like tyrosine kinase 3

FL:

Flt3 ligand

rhFL:

Recombinant human FL

HSC:

Hematopoietic stem cells

IL-3:

Interleukin-3

SCF:

Stem cell factor

G-CSF:

Granulocyte colony-stimulating factor

GM-CSF:

Granulocyte-macrophage colony-stimulating factor

M-CSF:

Macrophage colony-stimulating factor

PDGFR:

Platelet-derived growth factor receptor

VEGF:

Vascular endothelial growth factor

AML:

Acute myelogenous leukemia

CD:

Circular dichroism

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Acknowledgments

We are grateful to the Department of Ultrastructure at the Vrije Universiteit Brussel (Belgium) for access to CD-spectroscopy facilities. KV is a doctoral fellow of the Fund for Scientific Research-Flanders, Belgium (FWO). This work was supported by research grants from the FWO (G.0643.07) and Ghent University (BOF01Z01705) to S.N.S., and from the German Research Foundation (SFB 655 TP16) to C.T.

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Author notes

  1. Sevgi Ertugrul

    Present address: Department of Biology, Faculty of Science, Ankara Univeristy, 06100, Besevler, Ankara, Turkey

Authors and Affiliations

  1. Unit for Structural Biology, Laboratory for Protein Biochemistry and Biomolecular Engineering (L-ProBE), Ghent University, K.L. Ledeganckstraat 35, 9000, Ghent, Belgium

    Kenneth Verstraete, Sevgi Ertugrul & Savvas N. Savvides

  2. Medizinische Klinik und Poliklinik I, Universitätsklinikum Carl Gustav Carus der Technischen Universität Dresden, Fetscherstraße 74, 01307, Dresden, Germany

    Sina Koch & Christian Thiede

  3. Unit for Mass Spectrometry and Proteomics, Laboratory for Protein Biochemistry and Biomolecular Engineering (L-ProBE), Ghent University, K.L. Ledeganckstraat 35, 9000, Ghent, Belgium

    Isabel Vandenberghe, Maarten Aerts & Gonzalez Vandriessche

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  1. Kenneth Verstraete
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Correspondence to Savvas N. Savvides.

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Verstraete, K., Koch, S., Ertugrul, S. et al. Efficient Production of Bioactive Recombinant Human Flt3 Ligand in E. coli . Protein J 28, 57–65 (2009). https://doi.org/10.1007/s10930-009-9164-5

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