طراحی و ساخت کلون بیان کننده داروی ضد انعقادی دسیرودین (هیرودین) به شکل خارج سلولی در اشرشیا کلی

Background and purpose: Hirudin is a 65-66 amino acids polypeptide which is secreted as an anticoagulant compound from salivary glands of medical leech. This drug is a very potent inhibitor of thrombin and is so effective for arterial and venous thrombosis prevention. Therefore, it can compete with heparin. The aim of this study was to add a pelB signal peptide to pET-22b plasmid and to investigate the expression of recombinant hirudin in E.coli. Materials and methods: At first, the 66 nucleotic sequence of hirudin's gene was obtained and entered to a powerful vector (pET-22b). N_terminal His-tag was used for protein purification. We inserted pelB signal sequence at the begining of the gene to secrete protein to periplasmic region and culture medium. The expression of the target protein by origami (DE3) strain was then measured. Finally, the target protein was measured in periplasmic region, cytoplasm and culture medium. Results: The results showed that more protein was expressed in the periplasmic space and a small amount of protein secreted into the culture medium. Conclusion: Using the vectors capable of transferring the proteins to the periplasmic region, that is very favorable space for the formation of disulfide bonds and properly folding of the target protein, could decrease the production of inclusion bodies and increase the production of soluble and active proteins