title: Expression, purification and characterization of the extracellular domain of human Flt3 ligand in Escherichia coli.
abstract: Fms-like tyrosine kinase 3 ligand (Flt3 ligand, FL) is a cytokine that affects the growth, survival and/or differentiation of hematopoietic cells through the activation of specific tyrosine kinase receptors, and is potentially useful for in vitro HSC amplification. To express the extracellular domain of human Flt3 ligand (hFL(ext)) in Escherichia coli, we cloned hFL(ext) and constructed the recombinant expression vector pET32a-hFL(ext). hFL(ext) was successfully expressed in E. coli as a Trx fusion protein (Trx-hFL(ext)) under IPTG (isopropyl-beta-D: -thiogalactopyranoside) induction for 12 h at 30 degrees C. The Trx-hFL(ext) protein, expressed in inclusion bodies even at a low induction temperature, was successfully refolded and purified using dialysis and affinity chromatography. The purified hFL(ext) was biologically active and could effectively stimulate the proliferation of mouse bone marrow nucleated cells revealed by cell proliferation assay and colony forming assay. In addition, in synergize with G-CSF and TPO, recombinant purified hFL(ext) could stimulate ex vivo expansion of murine Lin(-)Sca-1(+)c-Kit(+) cells. Therefore, using the E. coli expression system and an affinity chromatography system, we successfully expressed, refolded, and purified a biologically active Trx-hFL(ext) protein which might be potentially useful for in vitro HSC amplification.
authors.affiliation: State Key Laboratory of Cancer Biology, Department of Medical Genetics and Developmental Biology, Fourth Military Medical University, Chang-Le Xi Street #17, 710032 Xi'an, China.
authors.forename: Xingcheng
authors.initials: X
authors.lastname: Zhao
authors.forename: Ping
authors.initials: P
authors.lastname: Zhang
authors.forename: Qiang
authors.initials: Q
authors.lastname: Liu
authors.forename: Fei
authors.initials: F
authors.lastname: He
authors.forename: Lei
authors.initials: L
authors.lastname: Feng
authors.forename: Hua
authors.initials: H
authors.lastname: Han
mesh_terms: D000818:Animals; D000067696:Ataxin-1; D000067528:Ataxins; D001854:Bone Marrow Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D003114:Colony-Forming Units Assay; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism*; D005110:Extracellular Space / Q000187:drug effects / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D017434:Protein Structure, Tertiary; D019009:Proto-Oncogene Proteins c-kit / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D013879:Thioredoxins / Q000378:metabolism; D013926:Thrombopoietin / Q000494:pharmacology
chemical_list: C000593749:ATXN1 protein, human; D000067696:Ataxin-1; D000067528:Ataxins; C000593750:Atxn1 protein, mouse; D004274:DNA, Recombinant; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; C084881:flt3 ligand protein; D016179:Granulocyte Colony-Stimulating Factor; D013879:Thioredoxins; D013926:Thrombopoietin; D019009:Proto-Oncogene Proteins c-kit
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