Abstract
Repeated use of L-asparaginase II enzyme, in the treatment of acute lymphoblastic leukaemia, is commonly needed because of the enzyme’s instability and relatively short half-life which leads to more serious side effects on patients. In the present study, we report on the cloning and expression of L-asparaginase from a thermotolerant strain of Escherichia coli (KH027) which was isolated from camel manure and could grow at 45°C. Expression of recombinant asparaginase was conducted by fusion asparaginase gene to pelB leader sequence and 6His residues at the C-terminus under the inducible T7 promoter in DH5α cells. Induction of the cells with 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at late log phase of growth resulted in 0.6-fold (2111 UI) higher to that obtained in early log phase induction (1319 UI) and 0.3-fold compared with mid log phase induction (1623 UI). The recombinant asparaginase protein was purified from the culture supernatant through nickel affinity chromatography. The apparent molecular weight of the tetramer enzyme was found to be ~141 kDa. Overall yield (87 mg/L) of the purified recombinant asparaginase was achieved at the shake flask level. The purified protein showed optimum activities at a temperature of 43°C and pH 6. The Km and Kcat parameters were 3.8 mM-1 and 2.92 × 103s−1, respectively. The enzyme retained around 57 and 30% of its initial activity after 30 and 60 min of incubation at 50°C, respectively. Recombinant L-asparaginase was evaluated for its antiproliferative effect in the leukemia cell lines of RS4; 11 and HL60 after 96 and 72 h of incubation. The doses of 100 μg/mL and time-response effect of 96 h caused a reduction value of 50% in cell viability of RS4. However, cell viability of 50% in the leukemic cells HL-60 was noticed with a concentration of 200 μg/mL with an incubation period of 72 h. In vitro antiproliferative results in the leukemia cell lines encourage for making in vivo investigation to increase the possibility of using this thermostable enzyme in leukaemia therapy. Key words: L-Asparaginase II, leukemia cell, recombinant asparaginase.
Highlights
Asparaginases are a cornerstone in the treatment protocols for acute lymphoblastic leukemia (ALL) (Pieters et al, 2011; Muller and Boos, 1998; Keating et al, 1993)
Treatment of ALL with L-asparaginase may lead to thrombotic complications and transient protein inhibition in the liver and pancreas (Nowak-Göttl et al, 2003) pancreatitis (Barry et al, 2007; Alvarez and Zimmerman, 2000; Kearney et al, 2009), diabetes, leucopoenia, neurological seizures are associated with the treatment course of ALL
Samples were collected at different stages of growth in order to determine the optimum conditions for asparaginase secretion
Summary
Asparaginases are a cornerstone in the treatment protocols for acute lymphoblastic leukemia (ALL) (Pieters et al, 2011; Muller and Boos, 1998; Keating et al, 1993). Depletion of the circulating pools of l-Asn byl-ASNase leads to the destruction of the tumour cells, since they are unable to complete protein synthesis (Kotzia and Labrou, 2007). Therapeutic response to those asparaginases of Erwiniachrysanthemi and E. coli rarely occurs without some evidence of toxicity (Duval et al, 2002; Pavelic et al, 2011) and hypersensitivity reactions (Panosyan et al., 2004; Woo et al, 2000; Larson et al, 1998). Treatment of ALL with L-asparaginase may lead to thrombotic complications and transient protein inhibition in the liver and pancreas (Nowak-Göttl et al, 2003) pancreatitis (Barry et al, 2007; Alvarez and Zimmerman, 2000; Kearney et al, 2009), diabetes, leucopoenia, neurological seizures are associated with the treatment course of ALL with L-asparaginase (Chabner and Loo, 1996; Duval et al, 2002)
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