Toward Safer Biotherapeutics: Expression and Characterization of a Humanized Chimeric L-Asparaginase in E. coli

Abstract

Acute lymphoblastic leukemia (ALL) is the most common cancer affecting children, making up about 80% of all acute leukemia cases in the pediatric population. While treatment with L-asparaginase (ASNase) has greatly improved survival rates, its bacterial origin often causes immune reactions in some patients, which can reduce how well the therapy works. To overcome this challenge, previous in silico studies designed a humanized chimeric ASNase by swapping out the predicted immunogenic parts of the bacterial enzyme with similar, less immunogenic segments from the human version-while keeping the enzyme's active site intact. In this study, the chimeric L-asparaginase designed was successfully cloned, expressed, and purified using the Escherichia coli Rosetta strain. The production conditions (37 °C, 0.01 mM IPTG, 2-4 h) were optimized, and we purified the enzyme in a single step with nickel-affinity chromatography. The enzyme's activity was confirmed in vitro, showing that it is possible to produce a functional humanized variant in a bacterial system. These results lay important groundwork for future research to assess the immune response and therapeutic potential of this novel chimeric enzyme.

Keywords: L-asparaginase (ASNase); acute lymphoblastic leukemia (ALL); chimeric; recombinant protein.

Figure 1

1% agarose gel electrophoresis of the amplification product of the humanized ASNase_Chimeric _E. coli-H. sapiens gene. The amplified bands of the gene of interest are indicated within a rectangle. Lanes S1, S2, and S3 are replicates of the sample and show a specific band of approximately 978 bp, corresponding to the expected size of the synthesized gene. Lane C represents the negative control without DNA template. A molecular weight marker (DNA ladder) is shown on the left, with the 1000 bp band used as a reference for estimating the product size.

Figure 2

Electrophoresis in 1% agarose gel of the PCR reaction of the colonies transformed with the recombinant plasmid containing the gene of interest. Bands are observed at a height of approximately 1000 bp corresponding to the plasmid transformed and amplified by the presence of the gene. Nothing is observed in the control or blank.

Figure 3

Humanized_E. coli-H. sapiens chimeric ASNase enzyme expression and activity assays after the induction process. (A) SDS-PAGE electrophoresis at 14% of the soluble(s) and insoluble fraction (i1, i2, and i3), and control for both fractions (Cs and Ci) for clones 2 and 4, using the E. coli BL21 strain as a host. (B) SDS-PAGE electrophoresis at 14% of the soluble(s) and insoluble fraction (i1, i2, and i3), and control for both fractions (Cs and Ci) for clones 2, 4, and 5, using the E. coli Rosetta (DE3) strain as a host. (C) Qualitative hydroxylamine enzyme activity assay of the soluble and insoluble fraction (i1, i2, and i3) using the E. coli Rosetta (DE3) strain as a host, E. coli Rosetta (DE3) strains modified with the empty vector as a control, and substrate without enzyme as the blank reagent. The brown coloration indicates L-asparaginase activity through the formation of a complex with hydroxylamine.

Figure 4

Response-surface plot representing the behavior of enzymatic activity in relation to IPTG concentration (mM), time (hours), and temperature (°C), generated using Design-Expert software. This software fixes one variable at a constant value while displaying the effects of the other two variables. In the presented graph, the Z-axis corresponds to enzymatic activity, while the X and Y axes represent IPTG concentration (mM) and time (hours), respectively. For this case, the temperature was fixed at 37 °C. The optimal conditions observed were an IPTG concentration of 0.01 mM and a time range of 2 to 4 h, where a maximum activity of approximately 2 U/mL is indicated in red on the plot. These values represent the optimal conditions for enzyme production. The data were fitted to a quadratic model, and ANOVA analysis indicated no significant differences (p > 0.05), confirming the adequacy of the model.

Figure 5

Chromatogram of ASNase_H_Q protein purification by histidine-tailed affinity chromatography using a nickel column on the AKTA Star. On the x-axis, in the ml column the volume is represented, and on the y-axis the absorbance (mAu) is represented. A peak between 44 and 50 mL, corresponding to the elution of the target protein, is observed. Elution was performed using a stepwise imidazole gradient from 100 to 500 mM; therefore, each step represents a specific concentration within the gradient. The protein eluted at 30%, corresponding to 150 mM imidazole.

Figure 6

12% SDS-PAGE electrophoresis of the soluble fractions collect of the chromatography. The protein of interest in the 35 kDa range is shown in red.

Figure 7

Western blot of ASNASA_H_Q chimeric protein. The black bands below M1 (sample 1) and M2 sample 2 represent the bands of the protein of interest run on electrophoresis and revealed with anti-His antibody from rabbit.