کلونینگ و تولید نوترکیب آنزیم گرانزیم M انسانی

Background and Objective: Granzyme M is a member of a granule serine proteases family, which is mainly expressed by cytotoxic T lymphocytes and natural killer cells. Granzyme M appears to be a potent inducer of tumor cell apoptosis. With respect to the importance of Granzyme M in the apoptosis, the aim of this work was the production of the biologically active enzyme. Materials and Methods: The cDNA fragment of active hGzm M, was cloned into bacterial expression vector pET21a (+). Recombinant plasmid was transformed into competent cells via heat shock method for protein expression. Induction condition was optimized to obtain high yield of recombinant protein and then protein expression analysis was done by SDS-PAGE. Produced inclusion bodies were dissolved in buffer solution containing Urea (8M) and then purified by Ni-Agarose Affinity Chromatography. The protease activity of granzyme M was assayed by spectrophotometric method, using casein as substrate. Results: Recombinant granzyme M was expressed as inclusion bodies in E. coli and expression of protein at 22°c with 1Mm IPTG during 5 hours provided the best protein expression level. Granzyme M was purified and refolded simultaneously by Ni-Agarose Affinity Chromatography using a concentration gradient of Urea. Results of the enzyme activity determination showed that the enzyme is active in the presence of casein and its specific activity is 71 U/mg. The activity of granzyme M showed that the enzyme was refolded properly. Conclusion: In this study a simple method was suggested to produce granzyme M and to determine the enzymatic activity of this enzyme which is involved in cancer cell apoptosis. This enzyme could be a potential target for anticancer drugs and the proposed method in this work facilitates studies on granzyme M.