L-Asparaginase is an enzyme successfully being used in the treatment of acute lymphoblastic leukemia， acute myeloid leukemia， and non-Hodgkin's lymphoma. However， some disadvantages still limit its full application potential， e.g.， allergic reactions， pancreatitis， and blood clotting impairment. Therefore， much effort has been directed at improving its performance. A popular strategy is to randomly conjugate L-asparaginase with mono-methoxy polyethylene glycol， which became a commercial FDA approved formulation widely used in recent years. To improve this formulation by PEGylation， herein we performed cysteine-directed conjugation of the L-asparaginase subunits to prevent dissociation-induce... More
L-Asparaginase is an enzyme successfully being used in the treatment of acute lymphoblastic leukemia， acute myeloid leukemia， and non-Hodgkin's lymphoma. However， some disadvantages still limit its full application potential， e.g.， allergic reactions， pancreatitis， and blood clotting impairment. Therefore， much effort has been directed at improving its performance. A popular strategy is to randomly conjugate L-asparaginase with mono-methoxy polyethylene glycol， which became a commercial FDA approved formulation widely used in recent years. To improve this formulation by PEGylation， herein we performed cysteine-directed conjugation of the L-asparaginase subunits to prevent dissociation-induced loss of activity. The recombinant cysteine conjugation sites were introduced by mutagenesis at surface-exposed positions on the protein to avoid affecting the catalytic activity. Three conjugates were obtained using different linear PEGs of 1000， 2000， and 5000 g/mol， with physical properties ranging from a semi-solid gel to a fully soluble state. The soluble-conjugate exhibited higher catalytic activity than the non-conjugated mutant， and the same activity than the native enzyme. The cysteine-directed crosslinking of the L-asparaginase subunits produced a higher molecular weight conjugate compared to the native tetrameric enzyme. This strategy might improve L-asparaginase efficiency for leukemia treatment by reducing glomerular filtration due to the increase in hydrodynamic size thus extending half-live， while at the same time retaining full catalytic activity.
