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Nan Fang Yi Ke Da Xue Xue Bao. 2012 Oct;32(10):1389-93.

Generation of streptavidin-tagged human-granulocyte macrophage colony-stimulating factor fusion proteins.

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1
Southern Medical University, Guangzhou, China. baili_2009ky@yahoo.cn

Abstract

OBJECTIVE:

To obtain streptavidin-tagged human granulocyte-macrophage colony-stimulating factor (SA/hGM-CSF) fusion protein and evaluate its bioactivity .

METHODS:

PET24a-6His-SA-L-hGM-CSF and PET24a-hGM-CSF-L-SA-6His plasmids were constructed and expressed in Rosetta (DE3) host bacteria to generate the fusion proteins. The two fusion proteins were refolded by gradient dialysis after Ni-NTA affinity chromatography and finally purified using DEAE-sepharose FF anion exchange chromatography. MTT method was used to evaluate the effect of SA/hGM-CSF fusion proteins in inducing the proliferation of human erythroleukemia cells (TF-1). The efficiency of the fusion proteins for surface modification of biotinylated MB49 tumor cells was evaluated by flow cytometry.

RESULTS:

The recombinant fusion proteins SA-hGM-CSF and hGM-CSF-SA were highly expressed in Rosetta (DE3) at about 20% of the total bacterial proteins, with a purity of about 96% after purification. The two fusion proteins exhibited bifunctional activities, namely the pro-proliferation effect on human erythroleukemia cells (TF-1) and SA-mediated high-affinity binding to biotinylated cell surfaces (with an anchoring modified rate of about 99%).

CONCLUSION:

SA/hGM-CSF bi-fusion proteins obtained in this study lays the groundwork for the development of cancer cell vaccines with surface modification by hGM-CSF.

PMID:
23076170
[Indexed for MEDLINE]
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