Research Of Cloning, Expression And Anti-cancer Activity On Bacterial Redox Protein: Azurin

The bacterial redox protein azurin belongs to the blue copperprotein family. Extraction of azurin from several kinds of bacteriumhas been reported, and azurin being researched in resent years isalmost all from Pseudomonas aeruginosa. In the past time, azurin wasextracted directly from all kinds of bacterium. These procedures werecomplicated and low efficient (about 1 mg pured protein per liter).What's more, much of these bacterium are pathogens which leadserious to environment pollution. With the development of molecularbiology, both amino acid sequence and base sequence of azurinbecome known to scientist, recombinant of azurin became reality. Inforeign labs, azurin was expressed with a signal peptide and secretedto the periplasmic space. Due to limit of periplasmic space itself, thisprocedure can obtain 2～5 mg pured protein per liter. Although it ismore efficient and safer than before, still it could not meet the need ofthe research now.We began with the azurin coding gene which was previouslyobtained from Pseudomonas aeruginosa and kept in pET-28 plasmid.The plasmid was transformed into E.coli DH5 strain, and re-extractedfrom the cultured bacterium. Azurin gene within Plasmid pET-28 wasexcised by Sal I and Nco I digestion and inserted into the Sal I site ofPlasmid pET-DB for expression. Resulting plasmid was transformedinto E.coli DH5 strain. More than 100 transformants were first selectedon LB plate with 50 mg/L kanamycin. Part of the positive clones werechosen for plasmid isolation. When the resulting plasmidpET-DB-azurin was digested by the endonucleases above and isolatedby electrophoresis, it raised a fragment of about 400 bp. The basesequence of the recombinant was correct according to the genebank.Then the plasmid was transformed into E.coli BL21(DE3) strain forexpression.The target protein is highly and solubly expressed according toSDS-PAGE. Analytical result by BANDSCAN software shows that theexpressed protein is 25 to 30 percent of total. Azurin is firstly purifiedby a Ni-NTA column. The purity of the resulting mixture is more than85%. Sephardex G75 gel is called for further purification. Azurinpurity is determined by SDS-PAGE and there is only one lane withmolecular weight of about 15 KD.This paper presents evidence that the recombinant azurin can notonly induce regression of tumor cells, but also HUVECs. Theregression is time dependent and concentration dependent. FlowCytometry analysis of azurin treated ACC-3 cells shows induction ofcell apoptosis. The effect of azurin on tube formation of HUVECsresults in no demonstratively different.The potential use of azurin or similar proteins with anticanceractivity in tumor therapy nevertheless has one significant problem. Asa protein, azurin will likely elicit antibody formation and be renderedineffective during long term treatment. It is thus important to usesmaller truncated derivatives of azurin that will elicit littlieimmunologic response in the body. In this paper, we digest azurin withtrypsin and find it could no longer cause regression of both tumor cellsand endothelium cells. Trypsin destroys the domain involving in itsanticancer activity. Further research on these digested sites might leadthe foundation of the functional domains of azurin.