Fig 1.
Sequence of the synthetic gene of L-asparaginase from Z. mobilis ATCC 31821.
The nucleotides in bold are the sequences of enzymes NcoI and XhoI. The nucleotides marked in gray are the sequence that encodes the six histidines. The underscored nucleotides are the sequence that encodes the enterokinase cleavage site.
Fig 2.
Phylogenetic tree of the different L-asparaginases from Z. mobilis, type II from E. coli and E. chrysanthemi, and type I from E. coli.
Note the grouping of the enzyme from Z. mobilis with the type II enzymes from E. coli and E. chrysanthemi, both of which have antileukemic activity.
Fig 3.
(a) Cell growth and activity of L-asparaginase using E. coli BL21 (DE3)/pET26b/ans (extracellular expression) induced after 203 minutes of culture; (b) growth and expression of the protein using E. coli BL21 (DE3)/pET28a/ans (intracellular expression) induced after 198 minutes of culture; (c) production of acetic acid over time; (d) specific activity of L-asparaginase over time as of induction of protein expression using the recombinant strains of L-asparaginase (E. coli BL21 (DE3)/pET26b/ans and E. coli BL21 (DE3)/pET28a/ans).
The results are the mean of the data, with error bars representing the standard deviation of triplicate values.
Table 1.
Cell concentration and enzyme activity of the cultures in bioreactors in LB medium at 37°C after 4 h induction with 0.55 mM IPTG.
Table 2.
Cell concentration and percentage of viable cells after treatment of Reh cells with recombinant L-asparaginase from Z. mobilis.
The cells were cultured in the presence and absence (control) of L-asparaginase 0.25 IU/mL and in the presence of a control without enzyme induction (negative control). Cell concentration and percentage of viable cells were determined by staining with PI and flow cytometry 48 h after treatment. The data are the mean ± standard deviation of the experiments in triplicate.
Fig 4.
Apoptotic effect of recombinant L-asparaginase from Z. mobilis on Reh cell line.
Cells were plated in triplicate and treated with recombinant L-asparaginase from Z. mobilis (L-AspZ) 0.25 IU/mL. They were incubated with Annexin V FITC and PI and analyzed by flow cytometry. Data represent the L-AspZ-induced increase in apoptotic cells, compared to the respective values observed in untreated control cultures and negative control cultures. The results are the median of the data, with error bars representing the standard deviation of triplicate values. *p ≤ 0.05.
Fig 5.
Effect of recombinant L-asparaginase from Z. mobilis on cell cycle distribution of Reh cell line.
Reh cells were treated with 0.25 IU/mL of L-asparaginase from Z. mobilis (L-AspZ), compared to the respective values observed in untreated control cultures and negative control cultures. The cells were stained with PI and DNA content was analyzed by flow cytometry. (a) Results are the median of data, with error bars representing the standard deviation of triplicate values. (b) The representative results of three independent experiments are shown. *p ≤ 0.05.
Fig 6.
Alteration in nuclear morphology by treatment with protein extract containing recombinant L-asparaginase from Z. mobilis after DAPI staining.
Representative images of Reh cells in fluorescence microscopy (1000x), (a) untreated control; (b) negative control and (c) the sample treated with protein extract containing recombinant L-asparaginase from Z. mobilis.
Fig 7.
Reduced proliferation of Reh cell line induced by Recombinant L-asparaginase from Zymomonas mobilis and commercial L-asparaginase.
Reh cells were treated with Recombinant L-asparaginase from Zymomonas mobilis and commercial L-asparaginase (0.00–1.00 IU/mL) and evaluated using the MTT assay. Data represent the L-asp-induced decrease in the percentage of viable cells compared to the respective values observed in control cultures. Results are the median of data, with error bars representing the standard deviation of triplicate values.
Table 3.
Clinical and laboratory characteristics of ALL patients.
Fig 8.
Effect of recombinant L-asparaginase from Z. mobilis on viability of ALL primary cells.
Cells were treated with recombinant L-asparaginase from Z. mobilis 0.1 IU/mL Viability was analyzed by flow cytometry 48h after treatment. Data represent the percentage viability of the treated cells relative to the respective values observed in parallel untreated control cultures. Results are the median of the data, with error bars representing the standard deviation of triplicate values.
Fig 9.
Cells from one ALL patient (PS4) treated with 0.0 IU/mL (control), 0.025 IU/mL, 0.05 IU/mL, 0.1 IU /mL, 0.5 IU/mL and 1.0 IU/mL recombinant L-asparaginase from Z. mobilis obtained from the E. coli BL21 (DE3)/pET26b/ans extracellular extract (L-AspZ) and stained with PI (X-axis).
The box-plots represent the median values of the total cell concentration, viable cell concentration, and dead cell concentration observed 48 h after the addition of L-asparaginase. The error bars represent the standard deviation of the triplicates. *p ≤ 0.05.