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Production and on-column re-folding of human vascular endothelial growth factor 165 in Escherichia coli

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Abstract

Vascular endothelial growth factors (VEGFs) are a family of proteins that promote angiogenesis and participate in a variety of physiological and pathological processes. In this work, the gene encoding the human VEGF isoform 165 (hVEGF165) was cloned into the expression vector pET32a (+) to construct a fusion expression plasmid that induced the thioredoxin (Trx) gene and transformed into Escherichia coli. The recombinant fusion protein TrxhVEGF165 was expressed optimally as inclusion bodies in the case of being cultivated for 4 h at 30°C and 1 mM IPTG concentration. The Trx-hVEGF165 was refolded and purified effectively from urea-solubilized inclusion bodies by the immobilized metal affinity chromatography. Released from the fusion protein by enterokinase cleavage and purified to homogeneity, the recombinant hVEGF165 (rhVEGF165) was biologically active as assessed by the human umbilicalvein endothelial cells (HUVECs) proliferation and the chicken chorioallantoic membrane (CAM) assay. The expression and in vitro refolding of rhVEGF165 resulted in production of an active molecule in a yield of 4.04 mg/L flask cultivation.

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Authors and Affiliations

  1. Department of Biological Sciences and the Research Institute for Basic Sciences, Hoseo University, Asan, 336-795, Korea

    Sun Kwon Bang, Young Sik Kim & In Seok Bang

  2. Department of Cosmetology, Hanseo University, Seosan, 356-706, Korea

    Byung Soo Chang

  3. Fusion Research Laboratory, Hoseo University, Asan, 336-795, Korea

    Cheol Beom Park

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  1. Sun Kwon Bang
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  2. Young Sik Kim
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Correspondence to In Seok Bang.

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Bang, S.K., Kim, Y.S., Chang, B.S. et al. Production and on-column re-folding of human vascular endothelial growth factor 165 in Escherichia coli . Biotechnol Bioproc E 18, 835–842 (2013). https://doi.org/10.1007/s12257-012-0829-0

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  • DOI: https://doi.org/10.1007/s12257-012-0829-0

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