Stabilization of L-asparaginase (ASNase) and Loading ASNase into Red Blood Cell by Fusion with 30Kc19 Protein of Bombyx mori

L-asparaginase (ASNase) from E. coli is one of the therapeutic enzymes of acute lymphoblastic leukemia (ALL). However, problems arise from direct injection of ASNase due to its short half-life and cause of immune response. Hence, to avoid these problems, loading ASNase into red blood cells (RBC) has been a popular method. In previous studies, we reported that the 30Kc19 protein from Bombyx mori enhanced enzyme stability. Moreover, we reported that the 30Kc19 protein has cell- penetrating property. In this work, we constructed pET-23a/ASNase-30Kc19 vector and overexpressed in E. coli system. We purified the ASNase-30Kc19 fusion protein by using fast protein liquid chromatography, and investigated the suppression of ASNase’s deactivation. By using spectrofluorometer, flow cytometer and confocal laser scanning microscopy, we investigated that Alexa Fluor® 488 labeled ASNase-30Kc19 was successfully loaded into the RBC. Surface morphology by scanning electron microscopy of the RBCs loaded with ASNase-30Kc19 also revealed that it was similar to that of the normal RBCs. Here, we demonstrated that by fusion with 30Kc19 cell-penetrating protein, the stability of ASNase was maintained and it also penetrated into RBCs successfully. Also, we verified the activity of ASNase-30Kc19-loaded RBCs compared with the therapeutic enzyme. To test the feasibility for the preliminary in vivo experiment, ASNase- 30Kc19- loaded RBCs will be tested in mouse model.