One‐step purification and kinetic properties of the recombinant l‐asparaginase from Erwinia carotovora

Abstract

ECAR-LANS, the recombinant L-asparaginase fromErwinia carotovora, is a prospective therapeutic enzyme for leukaemia treatment. An efficient and economical scheme was developed for the purification, cloning and expression in Eschericha coli of ECAR-LANS. More than 90 % purity, complemented with 72 % active enzyme recovery, was achieved with a single chromatographic purification step. The activity of purified L-asparaginase was 630 i.u./mg. The ECAR-LANS K m value was 98 × 10 −6 M for the main physiological substrate L-Asn and 3400 × 10 −6 M for L-Gln. ECAR-LANS was found to have low relative glutaminase activity (1.2 %) at physiological concentrations of L-Asn and L-Gln in blood. Kinetic studies of ECAR-LANS showed that the recombinant asparaginase combined the main advantages of Erw. chrysanthemi and E. coli L-asparaginases II, currently used in the treatment of acute lymphoblastic leukaemia.

Comparison between Two Erwinia carotovora L-Asparaginase II Constructions: cloning, Heterologous Expression, Purification, and Kinetic Characterization

“…There appears to be two minor components present in the AspMP preparation ( Figure 3). As previously reported (Krasotkina et al, 2004), these low-molecular-mass contaminants were probably proteolytic products of L-asparaginase II, which accumulated during storage of the enzyme solutions. Incidentally, no cocktail of protease inhibitors were employed in the purification process because their presence in a therapeutic protein must be avoided.…”

“…Previous reports cloned the DNA sequence of E. carotovora L-asparaginase II containing the signal sequence (Krasotkina et al, 2004;Kotzia and Labrou, 2005). Our aim was to compare expression and activity of two constructs of E. carotovora L-asparaginase II (with and without signal peptide) to optimize the process for large scale production.…”

“…However, no active protein yield was reported by these authors. Recombinant E. carotovora Lasparaginase II has been purified by a one-step purification proJournal of Microbial & Biochemical Technology -Open Access JMBT/Vol.2 Issue 1 tocol with 72% yield of active protein (Krasotkina et al, 2004), however the construct was protected by patent. Notwithstanding, the yield of active recombinant AspMP described here was 91% (Table 1), which is superior than reported by others (Krasotkina et al, 2004).…”

“…This is particularly important, since the glutaminase activity of therapeutic preparations of L-asparaginases II has been implicated in causing side effects such as serious liver disorders, acute pancreatitis, hyperglycemia and immunosuppression (Hawkins et al, 2004;Krasotkina et al, 2004). Interestingly, no report using the sequence of E. carotovora L-asparaginase II mature protein and recombinant DNA technology for its heterologous expression aiming at commercial production are available.…”

“…The assay employed to measure L-Asn and L-Gln hydrolysis catalyzed by homogeneous recombinant AspMP and AspSP proteins was the direct method described by Krasotkina et al (2004), with minor modifications. The reaction was carried out in a 1.5 mL quartz cuvette containing 198.9 nM AspSP or AspMP for Lasparaginase activity measurements and 662.9 nM AspSP or AspMP for L-glutaminase activity measurements, and one of the substrates (L-Asn or L-Gln) at varying concentrations (from 1 to 20 mM) in 0.02 M potassium phosphate buffer at pH 7.0.…”