Tehran University of Medical Sciences Acta Medica Iranica 0044-6025 61 4 2023 07 04 223 228 Mohammad Hassan Kheirandish Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran Mohammad Tollabi Department of Medical Biotechnology, School of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran Fatemeh Davami Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran Behnaz Rahmani Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran Negin Sadat Hashemi Aval Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran Javad Behroozi Department of Genetics and Advanced Medical Technology, Faculty of Medicine, AJA University of Medical Sciences, Tehran, Iran Hossein Zarei Jaliani Department of Medical Biotechnology, School of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran 2022 12 27 2023 05 07 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. https://acta.tums.ac.ir/index.php/acta/article/view/10492 https://acta.tums.ac.ir/index.php/acta/article/download/10492/5774