Goda, S.K., Rashidi, F.A.B., Fakharo, A.A. et al. Protein J (2009) 28: 435. doi:10.1007/s10930-009-9211-2
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.
Synthetic carboxypeptidase G2 CPG2 overexpression ADEPT Protein purification Synthetic Glucarpidase
Carboxypeptidase G2 (glucarpidase)
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis