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


CONCLUSIONS
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.
Southern Medical University, Guangzhou, China. baili_2009ky@yahoo.cn
Li
L
Bai
Zhiming
Z
Hu
Fei
F
Wang
Xiaoling
X
Xu
Chang
C
Xia
Liqin
L
Jin
Jinlong
J
Li
Jimin
J
Gao
D015415:Biomarkers; D019496:Cancer Vaccines / Q000096:biosynthesis; D045744:Cell Line, Tumor; D004224:Diterpenes / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis*; D006801:Humans; D050576:Membrane Fusion Proteins / Q000096:biosynthesis*; D010957:Plasmids; D019809:Streptavidin / Q000737:chemistry*
D015415:Biomarkers; D019496:Cancer Vaccines; D004224:Diterpenes; D050576:Membrane Fusion Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D019809:Streptavidin; C478098:dehydroandrographolide