Expression,purification and characterization of the extracellular domain of human Flt3 ligand in <Emphasis Type="Italic">Escherichia coli</Emphasis> |
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Authors: | Xingcheng Zhao Ping Zhang Qiang Liu Fei He Lei Feng Hua Han |
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Institution: | (1) 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; |
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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 (hFLext) in Escherichia coli, we cloned hFLext and constructed the recombinant expression vector pET32a-hFLext. hFLext was successfully expressed in E. coli as a Trx fusion protein (Trx-hFLext) under IPTG (isopropyl-β-d-thiogalactopyranoside) induction for 12 h at 30°C. The Trx-hFLext protein, expressed in inclusion bodies even at a low induction temperature, was successfully refolded and purified using
dialysis and affinity chromatography. The purified hFLext 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 hFLext 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-hFLext protein which might be potentially useful for in vitro HSC amplification. |
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