Bacterial co-expression of the α and β protomers of human l-asparaginase-3: Achieving essential N-terminal exposure of a catalytically critical threonine located in the β-subunit

• Co-expression of the two subunits α and β of human L-asparaginase-3 is reported.
• Fully active enzyme was obtained upon removal of N-terminal Met from the β-subunit.
• N-terminal Thr residue in the β-subunit is critical for catalysis by this hydrolase.
• Co-expression depends on vector type, position of tags, order of transcribed genes.

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 α and β) which remain non-covalently associated and constitute the catalytically active form of the enzyme. However, recombinant expression of full-length hASNase3 in Escherichiacoli results in only partial processing towards the active enzyme. We developed a co-expression system for the two subunits that allowed production of the β-subunit complexed to the α-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 β-subunit. The enzyme produced by this co-expression strategy is fully active, thus obviating the necessity of self-activation by slow autoproteolytic cleavage.

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