Functional overexpression and purification of a codon optimized synthetic glucarpidase (carboxypeptidase G2) in Escherichia coli.
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.
Goda
Sayed K
SK
College of Arts and Sciences, Qatar University, Doha, Qatar, sayed.goda@qu.edu.qa
Rashidi
Fatma A Baoumi
FA
Fakharo
Ameena A
AA
Al-Obaidli
Aisha
A
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D002479:Inclusion Bodies / Q000737:chemistry; D008969:Molecular Sequence Data; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015032:Zinc / Q000378:metabolism; D011623:gamma-Glutamyl Hydrolase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D011994:Recombinant Proteins; D011623:gamma-Glutamyl Hydrolase; D015032:Zinc