Tailoring structure-function properties of L-asparaginase: engineering resistance to trypsin cleavage

Georgia A Kotzia , Katerina Lappa , Nikolaos E Labrou (1)

1 Biotechnology

Bacterial L-asparaginases (L-ASNases) catalyze the conversion of L-asparagine to Laspartate and ammonia, and are widely used for the treatment of acute lymphoblastic leukemia (ALL). In the present study we describe an efficient approach, based on protein chemistry and protein engineering studies, for the construction of trypsinresistant PEGylated L-ASNase from Erwinia carotovora (EcaL-ASNase). Limited proteolysis of EcaL-ASNase with trypsin was found to be associated with a first cleavage of the peptide bond between Lys53 and Gly54, and then a second cleavage at the Arg206-Ser207 position of the C-terminal fragment, peptide 54-327, showing that the initial recognition sites for trypsin are Lys53 and Arg206. Site-directed mutagenesis of Arg206 to His followed by covalent coupling of methoxypolyethylene glycol succinate N-hydroxysuccinimide ester (mPEG-SNHS) to the mutant enzyme resulted in an improved modified form of EcaL-ASNase that retains 82% of the original catalytic activity, exhibits enhanced resistance to trypsin degradation, and has higher thermal stability compared to the wild-type enzyme.

Mots clés: Life Sciences

Georgia A Kotzia, Katerina Lappa, Nikolaos E Labrou. Tailoring structure-function properties of L-asparaginase: engineering resistance to trypsin cleavage. Biochemical Journal, 2007, 404 (2), pp.337-343. ⟨10.1042/BJ20061708⟩. ⟨hal-00478696⟩