Functional overexpression and purification of a codon optimized synthetic glucarpidase (carboxypeptidase G2) in Escherichia coli

Sayed K. Goda, Fatma A Baoumi Rashidi, Ameena A. Fakharo, Aisha Al-Obaidli

Research output: Contribution to journalArticle

7 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)435-442
Number of pages8
JournalProtein Journal
Volume28
Issue number9-10
DOIs
Publication statusPublished - Dec 2009
Externally publishedYes

Fingerprint

gamma-Glutamyl Hydrolase
Codon
Escherichia coli
Purification
Enzymes
Synthetic Genes
Enzyme Therapy
Genes
Prodrugs
Pseudomonas
Proteins
Recombinant Proteins
Methotrexate
Detoxification
Column chromatography
Names
Oncology
Chromatography
Zinc
Antibodies

Keywords

  • ADEPT
  • CPG2 overexpression
  • Protein purification
  • Synthetic carboxypeptidase G2
  • Synthetic Glucarpidase

ASJC Scopus subject areas

  • Biochemistry
  • Analytical Chemistry
  • Organic Chemistry
  • Bioengineering

Cite this

Functional overexpression and purification of a codon optimized synthetic glucarpidase (carboxypeptidase G2) in Escherichia coli. / Goda, Sayed K.; Rashidi, Fatma A Baoumi; Fakharo, Ameena A.; Al-Obaidli, Aisha.

In: Protein Journal, Vol. 28, No. 9-10, 12.2009, p. 435-442.

Research output: Contribution to journalArticle

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