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Expression, purification and characterization of the extracellular domain of human Flt3 ligand in Escherichia coli

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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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Abbreviations

FL:

Fms-like tyrosine kinase 3 ligand

HSC:

Hematopoietic stem cell

Trx:

Thioredoxin

hFLext :

The extracellular domain of human Flt3 ligand

IPTG:

Isopropyl-β-d-thiogalactopyranoside

G-CSF:

Granulocyte colony stimulating factor

TPO:

Thrombopoietin

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Acknowledgments

This study was supported by grants from the National Natural Science Foundation of China (30400079, 30425015, 30600544, 30801050), the Ministry of Science and Technology of China (2006AA02A111). The authors declare that they have no competing financial interests.

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Authors and Affiliations

  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

    Xingcheng Zhao, Ping Zhang, Qiang Liu, Fei He, Lei Feng & Hua Han

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  1. Xingcheng Zhao
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  2. Ping Zhang
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  3. Qiang Liu
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  4. Fei He
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  5. Lei Feng
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Correspondence to Ping Zhang or Hua Han.

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Zhao, X., Zhang, P., Liu, Q. et al. Expression, purification and characterization of the extracellular domain of human Flt3 ligand in Escherichia coli . Mol Biol Rep 37, 2301–2307 (2010). https://doi.org/10.1007/s11033-009-9724-y

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  • DOI: https://doi.org/10.1007/s11033-009-9724-y

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