Expression Of Human Tumor Necrosis Factor Alpha In Anabaena Sp. PCC7120 And Its Preliminary Purification

Human tumor necrosis factor α (TNF-α) is a cytokine produced in vivo by activated macrophages and monocytes. TNF-α is cytotoxic to several tumor cell lines in vitro and in vivo. TNF-α plays a significant role as local treatment for human cancer. Human TNF-α is an unglycosylated polypeptide, containing 157 amino acids, with a molecular mass of 17350Da. The active form of the molecular is a trimer. At present, TNF-α was mostly cloned and expressed in Escherichia coli. However, much expensive biochemical is required for cultivation of Escherichia coli. Furthermore, Escherichia coli contains toxic proteins and makes TNF-α 's purification complicated. Cyanobacteria are photosynthetic prokaryotes with many characteristics that make them a potentially ideal model for the study of a variety of biological processes. Since only carbon dioxide, sunlight and essential mineral are required for their cultivation, cyanobacterial bioprocesses are economic and clean. Cyanobacteria can be utilized as an alternative host for the recombinant DNA products. With the aim of applying recombinant cyanobacteria to anti-tumor cytokines production, we describe human TNF-α gene expression in the unicellular filamentous cyanobacterium, Anabaena sp. PCC7120 and its preliminary.In this study, the recombinant human TNF-α analog (rhTNF-α ) cDNA (approx. 0.7kb) was excised from PT-TNF plasmid by EcoR I and Xho I digestion. The extensions of the Xho I site was blunted by Klenow Polymerase. The rhTNF-α cDNA was inserted downstream of psbA promoter on the plasmid PRL439 to get the intermediate plasmid PRL-TNF (3.3kb). PRL-TNF was digested by EcoR I and Sal I to yield the fragment (0.8kb) containing psbA promoter and rhTNF-α cDNA. Then, the 0.8kb fragment was ligated to the shuttle vector PDC8 to get shuttle expressing vector PDC-TNF (9.9kb).The shuttle expressing vector PDC-TNF was transformed to unicellular filamentous cyanobacterium, Anabaena sp. PCC7120 by triparent conjugation and obtained genetically strain. Southern hybridization analysis showed that rhTNF-α...