Expression, purification and refolding of streptavidin-tagged human tumor necrosis factor-alpha fusion protein / 南方医科大学学报
Journal of Southern Medical University
;
(12): 412-415, 2009.
Article
in Chinese
| WPRIM
| ID: wpr-233775
ABSTRACT
<p><b>OBJECTIVE</b>To study the purification, refolding and bioactivity of streptavidin-tagged human tumor necrosis factor-alpha (SA-TNF-alpha) bi-functional fusion protein.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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%.</p><p><b>CONCLUSION</b>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.</p>
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Recombinant Fusion Proteins
/
Chemistry
/
Chromatography, Affinity
/
Tumor Necrosis Factor-alpha
/
Protein Folding
/
Streptavidin
/
Escherichia coli
/
Genetics
/
Metabolism
/
Methods
Type of study:
Prognostic study
Limits:
Humans
Language:
Chinese
Journal:
Journal of Southern Medical University
Year:
2009
Type:
Article
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