Expression of recombinant Escherichia coli L-asparaginase II, purification and characterization

Abstract

L-Asparaginase (ASNase) is an anti-cancer ("antineoplastic" or "cytotoxic") chemotherapy drug that is used for the treatment of acute lymphoblastic leukemia (ALL). An efficient and economical scheme was developed for over expression and rapid purification of the Escherichia coli enzyme. The gene encoding for the Escherichia coli L-asparaginase was PCR-amplified and cloned in pGEX-4T1 expression vector. The recombinant L-asparaginase was purified to homogeneity by affinity chromatography on glutathione Sepharose column. The recombinant enzyme had an apparent MW of 152 kDa and a Κ_m value of 12.5 μM for the main physiological substrate L-asparagine. The pI value was 5.6 while the turnover number (catalytic constant) was 1 x 10² s^-1 and the Κ_cat/Κ_m value (specificity constant) was 0.8 x 10⁷ M^-1s^-1 .

Key Word : L-asparaginase-II, Escherichia coli, cloning,overexpression affinity purification, acute lymphoblastic leukemia