Atefeh Khodakarami - Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
Bahareh Dabirmanesh - Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
Sedighe Asad - Department of Biotechnology, Faculty of Sciences, Tehran University, Tehran, Iran.
Khosro Khajeh - Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.

Introduction: Methotrexate is one of the most widely used chemotherapy agent that may cause kidney failure as a side effect. Carboxypeptidase (Glucarpidase) is a bacterial enzyme that could 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. Glucarpidase is marketed under the brand name of Voraxaze ($26,775 per 1000-unit vial). Materials and methods: In this research, we aimed to introduce an intein mediated purification with an affinity chitin-binding tag to simplify the purification of this valuable enzyme. Therefore, pET28a containing the carboxypeptidase G2 gene was extracted from bacteria and used as a templet in PCR reaction using primers containing Nde1 and SapI restriction sites. The PCR product was then digested and cloned into NdeI and Sap I restriction sites of an expression vector pTXB1 that encodes a thiol-cleavable C-terminal intein followed by a chitin-binding domain. The constructed recombinant plasmid was transformed into E. coli strain BL21 and expressed under different expression conditions. 0.1 mM IPTG, 25 °C and a 6 hr incubation provided the highest level of expression. Results: The results indicated that the purity of carboxypeptidase G2 and complete excision of the intein and CBD were confirmed by SDS-PAGE, while its proper folding was proved by circular dichroism and fluorescent emission studies. Conclusion: Therefore developing new methods to efficiently purify pharmaceutical proteins like carboxypeptidase G2 has attracted researchers’ attention. Self-cleaving intein mediated single column purification is one of these novel approaches. Self-cleaving inteins are modified forms of natural inteins that can excise and join only at one junction site.

Methotrexate, Carboxypeptidase G2, Intein, Purification