title: [Expression, purification and refolding of streptavidin-tagged human tumor necrosis factor-alpha fusion protein].
abstract: 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.
authors.affiliation: Institute of Biological Treatment, School of Biotechnology, Southern Medical University, Guangzhou, China. xucuixiang1129@163.com
authors.forename: Cui-Xiang
authors.initials: CX
authors.lastname: Xu
authors.forename: Zhi-Ming
authors.initials: ZM
authors.lastname: Hu
authors.forename: Jing-Long
authors.initials: JL
authors.lastname: Li
authors.forename: Ji-Min
authors.initials: JM
authors.lastname: Gao
mesh_terms: 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
chemical_list: D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D009532:Nickel; D019809:Streptavidin