Purification of a multimeric enzyme carboxypeptidase G2 by intein-mediated protein splicing

سال انتشار: 1395
نوع سند: مقاله کنفرانسی
زبان: انگلیسی
مشاهده: 833

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شناسه ملی سند علمی:

MPHBS01_158

تاریخ نمایه سازی: 22 آبان 1395

چکیده مقاله:

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.

نویسندگان

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.