Expression, Purification and Refolding of Recombinant Arginine Deiminase in Escherichia Coli and Its Activity as a Anti-tumor Enzyme

Abstract: Limited success has been achieved using amino acid degrading enzymes in cancer therapy. To date, only L-asparaginase is in clinical use for treating acute lymphoblastic leukemia and a few sub-types of non Hodgkin's lymphoma. The therapeutic effectiveness of amino acid deprivation therapy in cancer treatment depends not only on the enzymatic properties, such as specific activity, optimum pH and stability, but more importantly on the susceptibility of the target cells to the lack of the specific amino acid. However, asparaginase treatment is sometimes accompanied by serious side-effects including anaphylactic shock, coagulopathies as well as liver and pancreatic toxicity. 1 In addition, in some patients, the enzyme appears to be ineffective. These properties outline the need for alternative treatmentsArginie deiminase(ADI),which catalyzes the hydrolysis of L-arginine to L-citrulline and ammonia,exists in some microorganisms.L-arginine is an essential nutrient for most mammalian cells and is utilized as a major non-glycolytic metabolic energy source by Mycoplasma.Arginine deiminase ( ADI, EC 3.5.3.6 ) , isolated from the mycoplasma-contaminated cell culture, has been demonstrated that has important effects to inhibit tumor cell growth in vitro and has anti-angiogenic activity . In this study, we cloned a recombinant ADI (rADI) by PCR and then expressed in Escherichia coli. The rADI was purified with ion-exchange chromatogram to homogeneity. After renatured, the rADI was found have similar molecular weight (~46KD) and enzyme activity of converting L-arginine to citrulline (enzyme activity is 38 U/mg) as the native ADI has. Based on the results from proteolysis assay, we found that rADI exhibited more resistant to trypsin digestion as compared with the wild-type. A longer half-life was also found in rADI. Further research indicated that this rADI has an inhibitory effect on the growth of HepG2 cells and the anti-angiogenic activity can be validated by the inhibition of migration of HepG2 cells. There are many laboratories that have reported the arginine deiminase purification methods, but these purification methods are deficient very much, such as the complicated and unreasonable procedure. The more purification steps, the more loss of protein activity, because every step will inevitably cause the activity loss, moreover, each purification step will consume time, so that the purification procedure will on the relatively high cost. Purification of the irrational, Commonly, the recombinant arginine deiminase proteins are inactive and inclusion bodies in cells, and it is very critical to renature. The reported purification methods are generally diluted renature followed by purification. Because of the low concentration protein, dilution of rehabilitation after the very low concentration of protein, it is easy to cause loss of protein activity. Our experiment used only one purification step and then protein was renatured with dialysis in the appropriate buffer ,so that we not only can save time, cost, but also can maintain a maximum of protein activity.Cancer is increasingly a threat to human health of major diseases, and people are looking for all kinds of anti-cancer approach. There are many reports about the anti-cancer activity and mechanism about arginine deiminase, and it is believed that this anti-tumor cells will become a hot research. In our experiment, we have developed a fast, efficient method that express and purify arginine deiminase, and its anti-cancer activity has been verified…
Key words: Argimine deiminase; Inhibitory mechanism; Tumor cell growth; Mycoplasma

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