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

Content Provider	Springer Nature Link
Author	Goda, Sayed K. Rashidi, Fatma A. Baoumi Fakharo, Ameena A. Al obaidli, Aisha
Copyright Year	2009
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 Ni$^{2+}$ charged column chromatography. The synthetic recombinant glucarpidase expressed within this system was biologically active and zinc dependant. Our study showed that Mg$^{2+}$ as well as Mn$^{2+}$ ions inhibit the activity of the recombinant enzyme.
Starting Page	435
Ending Page	442
Page Count	8
File Format	PDF
ISSN	15723887
Journal	The Protein Journal
Volume Number	28
Issue Number	9-10
e-ISSN	15734943
Language	English
Publisher	Springer US
Publisher Date	2009-11-13
Publisher Place	Boston
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Synthetic carboxypeptidase G2 CPG2 overexpression ADEPT Protein purification Synthetic Glucarpidase Animal Anatomy / Morphology / Histology Organic Chemistry Biochemistry Bioorganic Chemistry
Content Type	Text
Resource Type	Article
Subject	Organic Chemistry Analytical Chemistry Biochemistry Bioengineering

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