[Expression, purification and refolding of streptavidin-tagged human tumor necrosis factor-alpha fusion protein].
OBJECTIVE
To study the purification, refolding and bioactivity of streptavidin-tagged human tumor necrosis factor-alpha (SA-TNF-alpha) bi-functional fusion protein.


METHODS
SA-TNF-alpha fusion protein was expressed in BL21(DE3) host bacteria, purified using Ni-NTA affinity chromatography and refolded by dilution and dialysis followed by identification using Western blotting. The effect of SA-TNF-alpha fusion protein against L929 cells was evaluated by MTT assay. Flow cytometry was used to analyze the surface modification of biotinylated MB49 tumor cells by SA-TNF-alpha fusion protein.


RESULTS
Recombinant SA- TNF-alpha fusion protein was expressed in BL21(DE3) at about 30% of total bacterial protein, with a purity of about 95% after purification. The SA-TNF-alpha fusion protein existed as dimmers, tetramers and higher order structures after refolding. The fusion protein exhibited a bi-functionality by inhibiting L929 cells and SA-mediated high-affinity binding to biotinylated cell surfaces, with an anchor modification rate of above 90%.


CONCLUSIONS
The dimmers, tetramers and higher order structures of the obtained SA-TNF-alpha fusion protein all exhibit a bi-functionality, and may serve as a potential candidate therapeutic agent for tumors.
Xu
Cui-Xiang
CX
Institute of Biological Treatment, School of Biotechnology, Southern Medical University, Guangzhou, China. xucuixiang1129@163.com
Hu
Zhi-Ming
ZM
Li
Jing-Long
JL
Gao
Ji-Min
JM
D002846:Chromatography, Affinity / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009532:Nickel; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D019809:Streptavidin / Q000096:biosynthesis* / Q000235:genetics; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000235:genetics
D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D009532:Nickel; D019809:Streptavidin