title: Efficient production of bioactive recombinant human Flt3 ligand in E. coli.
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.
authors.affiliation: Unit for Structural Biology, Laboratory for Protein Biochemistry and Biomolecular Engineering (L-ProBE), Ghent University, K.L. Ledeganckstraat 35, 9000, Ghent, Belgium.
authors.forename: Kenneth
authors.initials: K
authors.lastname: Verstraete
authors.forename: Sina
authors.initials: S
authors.lastname: Koch
authors.forename: Sevgi
authors.initials: S
authors.lastname: Ertugrul
authors.forename: Isabel
authors.initials: I
authors.lastname: Vandenberghe
authors.forename: Maarten
authors.initials: M
authors.lastname: Aerts
authors.forename: Gonzalez
authors.initials: G
authors.lastname: Vandriessche
authors.forename: Christian
authors.initials: C
authors.lastname: Thiede
authors.forename: Savvas N
authors.initials: SN
authors.lastname: Savvides
mesh_terms: D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D008565:Membrane Proteins; D011994:Recombinant Proteins; C084881:flt3 ligand protein; D020929:Mitogen-Activated Protein Kinase Kinases