Cloning, expression, purification and characterisation of Erwinia carotovora L-asparaginase in Escherichia coli

Abstract

Background: For the past 30 years, bacterial L-asparaginases have been used as therapeutic agents in the treatment of acute childhood lymphoblastic leukemia. It is found in a variety of organisms such as microbes, plants and mammals. Their intrinsic low-rate glutaminase activity, however, causes serious side-effects, including neurotoxicity, hepatitis, coagulopathy and other dysfunctions. Erwinia carotovora asparaginase shows decreased glutaminase activity, so it is believed to have fewer side-effects in leukemia therapy. Our aim was to clone, express, purify and characterize E. carotovora asparaginase.

Materials and methods: L-asparaginase from E. carotovora NCYC 1526 (ErA) was cloned and expressed in Escherichia coli strain BL21 (DE3). The enzyme was purified to homogeneity by affinity chromatography. Various conditions were tested to maximize the production of recombinant asparaginase in E. coli.

Results: A new L. asparaginase from E. carotovora NCYC 1526 (ErA) was successfully cloned, expressed and purified in E. coli BL21 (DE3). The specific activity of the enzyme was 430 IU/mg.

Conclusion: The results of the present work form the basis for a new engineered form of ErA for future therapeutic use, which could be extended with crystallographic studies.

Keywords: Characterization; ErA; Erwinia carotovora; Escherichia coli BL21 (DE3); L-asparaginase; cloning; purification.

Figure 1

Electrophoresis in 0.75% agarose gel of restriction endonuclease digestion of polymerase chain reaction product (a; 1.05 kb), pET22b (b; 5.4 kb) using BamHI and NdeI and recombinant construct (c; 6.5 kb) using just BamHI. Marker Hyperlader I from Bioline

Figure 2

Western blot analysis of samples to find optimal induction conditions, probed with His-tag antisera. Lane 1, uninduced sample, Lane 2-5, induced with 0.1, 0.5, 1 and 2 mM isopropyl β-D-1- thiogalactopyranoside

Figure 3

Western blot analysis of the samples for small scale purification, probed with His-tag antisera. Lane 1: Total lysate, Lane 2: Flow through sample from the column, Lane 3: The purified protein

Figure 4

The change of absorbance in collected samples from the fraction collector

Figure 5a and b

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12% gel) with the purified L-asparaginase to determine the presence of protein in the eluted samples. The gel was stained with Coomassie Brilliant Blue R-250. M: molecular size marker. Lane 1: sample of loaded crude supernatant to the column, Lane 2: Column effluent (Flow through), Lanes 3-7: Eluted fractions from tubes 58, 65, 69, 72 and 75. (a) Lanes 8-14: Samples of eluted fractions from tubes 77, 80, 82, 85, 88, 91 and 95 (b)

Figure 6

The change of absorbance in collected concentrated samples from fraction collector

Figure 7

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12% gel) with the purified L-asparaginase to determine the presence of protein in the eluted samples from the concentrated samples (Tubes 23, 24,25,26,27 and 28, respectively)