Effect of various additives on carboxypeptidase G2 G2 thermal stability

Carboxypeptidase is a bacterial dimeric zinc-dependent exopeptidase (derived from Pseudomonas sp. strain RS-16) that hydrolyze the C-terminal glutamate moiety from folic acid and its analogues, such as methotrexate1. High-dose methotrexate has been particularly useful in the treatment of leukemias and lymphomas. Because methotrexate is largely excreted by the kidneys, this can greatly potentiate tissue damage. Toxic levels of blood methotrexate can be rapidly and effectively decreased by intravenous administration of carboxypeptidase G2. However, the utilization of carboxypeptidase G2 as therapeutics is notably restricted due to its thermal instability2. Therefore, in this study, we decided to examine the enzyme stability in different additives such as glycerol, sorbitol, trehalose, sucrose and fructose monohydrate. For this reason pET21a containing SUMO-carboxypeptidase G2 gene was transformed into E.coli strain BL21 (DE3). Carboxypeptidase G2 was then expressed in LB medium induced with 1 mM IPTG at 20°C. The recombinant protein was over-expressed as soluble protein and SUMO tag was removed by SUMO protease. The purity of carboxypeptidase G2 and its complete excision of the SUMO tag were confirmed by SDS-PAGE. The enzyme activity was analyzed using methotrexate as a substrate. The Km and Vmax values for this enzyme, respectively was 0.012 mM and 0.016 μmol/min. Irreversible thermoinactivation of carboxypeptidase G2 has been evaluated at 50°C in the presence and absence of glycerol, sorbitol, and sucrose. Among examined additives, sorbitol had the most thermostabilizing effect.

Keywords:

Carboxypeptidase G2 , SUMO tag , Expression , Thermal stability , Trehalose