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

METHODSSA-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.

RESULTSRecombinant 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%.

CONCLUSIONThe 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.