Application of repeated aspartate tags to improving extracellular production of Escherichia colil-asparaginase isozyme II

• l-asparaginase isozyme II (AnsB) from E. coli is a therapeutic agent against acute lymphoblastic leukemia.
• Repeated aspartate tags were devised to express AnsB extracellularly in E. coli.
• A five-aspartate tag was optimal for extracellular production of AnsB.
• 40.8 U/ml of AnsB was obtained in a batch fermentation.
• AnsB was secreted into medium via the E. coli general secretion pathway.

Asparaginase isozyme II from Escherichia coli is a popular enzyme that has been used as a therapeutic agent against acute lymphoblastic leukemia. Here, fusion tag systems consisting of the pelB signal sequence and various lengths of repeated aspartate tags were devised to highly express and to release active asparaginase isozyme II extracellularly in E. coli. Among several constructs, recombinant asparaginase isozyme II fused with the pelB signal sequence and five aspartate tag was secreted efficiently into culture medium at 34.6 U/mg cell of specific activity. By batch fermentation, recombinant E. coli produced 40.8 U/ml asparaginase isozyme II in the medium. In addition, deletion of the gspDE gene reduced extracellular production of asparaginase isozyme II, indicating that secretion of recombinant asparaginase isozyme II was partially ascribed to the recognition by the general secretion machinery. This tag system composed of the pelB signal peptide, and repeated aspartates can be applied to extracellular production of other recombinant proteins.