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Abstract

L-asparaginases hydrolyze L-asparagine to L-aspartic acid and ammonia. Enzymes of bacterial origin are used as therapeutic agents for the treatment of acute lymphoblastic leukemia. Recently, the structure of a human homolog, hASNase3, which possesses L-asparaginase activity, was solved setting the basis for the development of an anti-leukemic protein drug of human origin. Being an N-terminal hydrolase, hASNase3 undergoes intramolecular self-cleavage generating two protomers (subunits alpha and beta) which remain non-covalently associated and constitute the catalytically active form of the enzyme. However, recombinant expression of full-length hASNase3 in Escherichia coli results in only partial processing towards the active enzyme. We developed a co-expression system for the two subunits that allowed production of the beta-subunit complexed to the a-subunit such that the N-terminal methionine is removed by endogenous methionine aminopeptidase to expose the catalytically essential threonine residue at the N-terminus of the beta-subunit. The enzyme produced by this co-expression strategy is fully active, thus obviating the necessity of self-activation by slow autoproteolytic cleavage.