الفهرس 
IntroductionOnly 14 pages are availabe for public view
 |  | from | 30 |  |  |
| | | from | 30 | | |
Abstract
L-Asparaginase (L-ASNase EC 3.5.1.1) is considered as an important biopharmaceutical drug enzyme in the treatment of childhood acute lymphoblastic leukemia (ALL). In the present study, Pyrocoecus furiosus L-ASNase gene was cloned into pET26b (+), expressed in E. coli BL21(DE3) pLysS. and purified to homogeneity using Ni2+ chelated Fast Flow Sepharose resin with 5.7 purification fold and 23.9% recovery. The purified enzyme exhibited a molecular weight of -33,660 Daltons on SDS-PAGE and showed maximal activity at 50 °C and pH 8.0. It retained 98.3% and 60.7% initial activity after 60 min at 37 °C and 50 °C, respectively.
The recombinant enzyme showed highest substrate specificity towards L-ASNase substrate, while no detectable specificity was observed for L-glutamine, urea, and acrylamide at 10 mM concentration. The Km and Vrnax of the purified recombinant enzyme as calculated using Lineweaver-Burk plot were determined to be 1.623 mM and 105 pmol min-1 mg-1, respectively.
The effect of sulfate and chloride metal ions, and reducing agents was investigated showing both KC1 and NaCl enhance the L-ASNase activity at a concentration of ImM. the effect of free amino acid L. glycine on the activity of P. furiosus purified recombinant L. ASNase was investigate also and significantly showed enhancement of the L. ASNase activity, the L. ASNase activity was higher at 250mM glycine than at 500mM after 3 and 48 h of incubation period at room temperature and at 4 °C (280% and 260% respectively).
The hemolytic effect of P. furiosus recombinant L. ASNase did not exhibit any hemolysis on human blood. Moreover, quantitative hemolysis test also showed same result on erythrocytes as compared to a positive control that was a lysis solution containing 1%SDS and 0.4 N NaOH. Serum and trypsin half-lives of recombinant P. furiosus L-ASNase were also investigated and showed that recombinant L-ASNase retained 50% of its initial activity after 55.5% incubation period with trypsin, while it retained 37.56% after 80 min. On the contrary, an incubation with human serum at 37 °C for 2.5 h resulted in 50% reduction of L-ASNase activity.
In vivo study on rats received acute dose of recombinant P. furiosus L. ASNase did not show significant differences on hepatic enzymes, albumin, cholesterol, and triglycerides. The activity detected after 2 h in animal group received 5700 IU decreased after 8 h to 3.535% from the original activity.
Human leukemia cell line THP-1 treated with recombinant L-ASNase showed significant morphological changes, and the IC50 of the purified enzyme was found to be 0.8 IU. Moreover, the purified recombinant L-ASNase induced cytotoxic effects on lung adenocarcinoma A549 and colorectal adenocarcinoma Caco-2 cell lines with IC50 of 1.78 IU and 30 IU, respectively., Thus, the aims of the proposed protocol are to produce and to develop microbial l* asparaginases. So. in addition to the commercially available l- asparaginases we propose to seek for other asparaginases from different prokaryotes and eukaryotes sources in an attempt to reduce or abolish the immune response.