Volume 26, Issue 10 (12-2019)                   RJMS 2019, 26(10): 8-18 | Back to browse issues page

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Aalizade R, Kardan N, Khodakarami A, Khajeh K, Dabirmanesh B. Cloning, expression and Characterization of Carboxypeptidase G2 Enzyme from E.coli. RJMS 2019; 26 (10) :8-18
URL: http://rjms.iums.ac.ir/article-1-5093-en.html
Tarbit Modares University, Tehran, Iran , dabirmanesh@modares.ac.ir
Abstract:   (3379 Views)
Background: Methotrexate is one of the most widely used chemotherapeutic agents that may cause kidney failure as a side effect. Carboxypeptidase G2 (Glucarpidase marketed under the brand name of Voraxaze) is a bacterial enzyme that can convert methotrexate to its inactive metabolites and provides an alternative non-renal pathway for methotrexate elimination in patients with renal dysfunction during high-dose methotrexate treatment.
Methods: In this research, carboxypeptidase G2 was synthetized in pUC 57 vector. Then it was subcloned into pET28a between NdeI and XhoI restriction sites. Recombinant vector was transformed into E. coli BL21 and its expression was examined in various conditions.   
Results: The optimum expression of recombinant protein was obtained at the concentration of 0.5 mM IPTG, 25 ° C, 6 h. Then the active enzyme was purified by Nickle affinity chromatography. Optimal pH and temperature of enzyme was 7 and 25 ˚C respectively. The Km and Vmax for methotrexate were 24 µM and 0.005431 µmol/min.  
Conclusion: The present study was conducted to produce recombinant carboxypeptidase G2 and to improve its production efficiency. In future, this enzyme could be a potential candidate for preventing chemotherapy side effects.
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Type of Study: Research | Subject: Clinical Biochemistry

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