Bacterial Expression of TMTP1-Fused L-Asparaginase for Targeting Leukemia and Metastatic Tumor Cells
L-asparaginase is recognized as a first-line anticancer drug for
acute lymphoblastic leukemia (ALL); however, low-substrate specificity
and exhibiting glutaminase activity cause various off-target toxicities
on normal cells. In the following study, we functionalized wild-type
asparaginase with the TMTP1 targeting peptide which specifically targets
a variety of hematological and metastatic cancer cells. The peptide
sequence was genetically added to the N-terminal end of the asparaginase
using the restriction endonuclease- free cloning method. Wild-type and
engineered asparaginases were expressed in E. coli and purified by
Nickel affinity chromatography column. The in vitro activity of both
types of enzymes was evaluated by Nessler’s method. The sequencing
results showed that the TMTP1 sequence was added in the correct frame to
the asparaginase. Wild-type and TMTP1-fused asparaginases were produced
in a soluble state with the specific activity of 172 U/mg and 153 U/mg,
respectively. The evidence from this study suggests that TMTP1-fused
asparaginase could preserve its solubility and activity compared to the
wild-type species and can be proposed for future research in anticancer
therapies.
Keywords: L-asparaginase; TMTP1 targeting peptide; Acute lymphoblastic leukemia; Restriction endonuclease-free cloning