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Author Goda, Sayed K.en_US
Author Rashidi, Fatma A. Baoumien_US
Author Fakhroo, Amina A.en_US
Author Al-obaidli, Aishaen_US
Available date 2009-12-24T08:37:37Zen_US
Publication Date 2009-11-13en_US
Publication Name The Protein Journal
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), 435–442en_US
ISSN 1572-3887 (Print)en_US
ISSN 1573-4943 (Online)en_US
URI http://dx.doi.org/10.1007/s10930-009-9211-2en_US
URI http://hdl.handle.net/10576/10446en_US
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 enzymeen_US
Language enen_US
Publisher Springer USen_US
Subject Synthetic carboxypeptidase G2en_US
Subject CPG2 overexpressionen_US
Subject ADEPTen_US
Subject Protein purificationen_US
Subject Synthetic Glucarpidaseen_US
Title Functional Overexpression and Purification of a Codon Optimized Synthetic Glucarpidase (Carboxypeptidase G2) in Escherichia colien_US
Type Articleen_US


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