E coli asparaginase

Culture media ingredients, tryptone, and yeast extract were from Difco Laboratories, India. Tris buffer, glycine, IPTG, sodium dodecyl sulfate, PMSF, and deoxycholic acid were from Amresco, USA. Ammonium persulfate, acrylamide, bis-acrylamide, E. coli asparaginase, L-arginine and Urea were from Sigma Chemicals, USA. DEAE-sepaharose and S-200 gel filtration matrix was from GE Healthcare, Sweden. TEMED, EDTA, bromophenol blue from Biorad, USA. Coomassie brilliant blue R-250 and ampicillin from USB Corporation, Cleveland, Ohio. Glucose, NaCl, Nessler's reagent, and other chemicals were from Qualigen, India.

Leukemia cell lines and PBMCs were seeded in 96-well plates at a density of 1x10⁴ cells/well, HDFa cells were seeded at a density of 5x10³cells/well. All the cells were incubated with different concentrations of purified recombinant proteins for 24h. Microscopy was performed on a Leica DMI400B automated Inverted Microscope, images were captured using a Leica DFC 350 FX camera (10X Magnification) and acquired by Leica Application Suite Software (Version 2.8.1).
Cells were then treated with the CCK8 reagent (Sigma), following manufacturer's instructions. Cell viability was evaluated by determining the concentration of WST-8-formazan, a compound with a typical absorbance at 450 nm, which formation is directly proportional to the number of living cells. Absorbance was read by using a microplate reader (Thermo Fisher). The viability of cells was also evaluated by the Trypan Blue exclusion assay. Experiment of comparison of cytotoxic activity between GST-ASPG and bacterial Asparaginase was performed by using E. Coli asparaginase (Sigma). Cytotoxicity of GST-ASPG on leukemia cells was also evaluated in the presence of D-asparagine (200 mM), L-asparagine (50 mM) and PAF (10μM), using the CCK8 assay.