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

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

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dc.contributor.author Goda, Sayed K.
dc.contributor.author Rashidi, Fatma A. Baoumi
dc.contributor.author Fakhroo, Amina A.
dc.contributor.author Al-obaidli, Aisha
dc.date.accessioned 2009-12-24T08:37:37Z
dc.date.available 2009-12-24T08:37:37Z
dc.date.issued 2009-11-13
dc.identifier.citation Volume 28, Numbers 9-10 / December, 2009 en_US
dc.identifier.issn 1572-3887 (Print)
dc.identifier.issn 1573-4943 (Online)
dc.identifier.uri http://dx.doi.org/10.1007/s10930-009-9211-2
dc.identifier.uri http://hdl.handle.net/10576/10446
dc.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
dc.language.iso en en_US
dc.publisher Springer Netherlands en_US
dc.subject Synthetic carboxypeptidase G2 en_US
dc.subject CPG2 overexpression en_US
dc.subject ADEPT en_US
dc.subject Protein purification en_US
dc.subject Synthetic Glucarpidase en_US
dc.title Functional Overexpression and Purification of a Codon Optimized Synthetic Glucarpidase (Carboxypeptidase G2) in Escherichia coli en_US
dc.type Article en_US

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