Functional Overexpression and Purification of a Codon Optimized Synthetic Glucarpidase (Carboxypeptidase G2) in Escherichia coli

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contributor.author Goda, Sayed K. en_US
contributor.author Rashidi, Fatma A. Baoumi en_US
contributor.author Fakhroo, Amina A. en_US
contributor.author Al-obaidli, Aisha en_US
date.accessioned 2009-12-24T08:37:37Z en_US
date.available 2009-12-24T08:37:37Z en_US
date.issued 2009-11-13 en_US
identifier.citation Goda, S., Rashidi, F. B., Fakharo, A., & Al-obaidli, A. (2009). Functional Overexpression and Purification of a Codon Optimized Synthetic Glucarpidase (Carboxypeptidase G2) in Escherichia coli. The Protein Journal, 28(9-10), 435–442 en_US
identifier.issn 1572-3887 (Print) en_US
identifier.issn 1573-4943 (Online) en_US
identifier.uri http://dx.doi.org/10.1007/s10930-009-9211-2 en_US
identifier.uri http://hdl.handle.net/10576/10446 en_US
description.abstract Glucarpidase (former name: carboxypeptidase G2, or CPG2) is a bacterial enzyme that is widely used in detoxification of the cytotoxic drug, methotrexate, and in Antibody Directed Enzyme Prodrug Therapy for cancer treatment. The glucarpidase gene of Pseudomonas sp. strain RS-16 was previously cloned in E coli, but expresses at a level that is approximately 100-fold lower than in the native strain. In this study, a synthetic gene coding for glucarpidase was codon-optimised and synthesized for maximum expression in E. coli using the vector pET28a. Our work indicated that the enzyme was expressed to ~60% of the total host protein and that purification of the recombinant His-tagged protein could be achieved in a single step by Ni2+ charged column chromatography. The synthetic recombinant glucarpidase expressed within this system was biologically active and zinc dependant. Our study showed that Mg2+ as well as Mn2+ ions inhibit the activity of the recombinant enzyme en_US
language.iso en en_US
publisher Springer US en_US
subject Synthetic carboxypeptidase G2 en_US
subject CPG2 overexpression en_US
subject ADEPT en_US
subject Protein purification en_US
subject Synthetic Glucarpidase en_US
title Functional Overexpression and Purification of a Codon Optimized Synthetic Glucarpidase (Carboxypeptidase G2) in Escherichia coli en_US
type Article en_US


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