en بیوشیمی Biochemistry پژوهشي Research <div style="text-align: justify;"><strong>Background:</strong> DAB₃₈₉IL-2 is considered a fusion immunotoxin and it is used for the CTCL therapy. DAB₃₈₉IL-2 includes of two distinct portions; the catalytic domain of diphtheria toxin and IL-2. DAB₃₈₉IL-2 duo to the presence of a free cysteine residue (Cys 513 in IL-2 part) is prone to unwanted intramolecular and intermolecular disulfide bonds formation and aggregation problems. Aggregation is considered as the most common physical instability. PEGylation is an effective approach to increase the stability and half-life of therapeutic proteins.<br> <strong>Materials and methods:</strong> In this study, the PEGylation of recombinant DAB₃₈₉IL-2 was performed by mPEG-vinylsulfone, through partial denaturation condition at 4 ⁰C for 24 h. To confirm the PEGylation reaction, SDS-PAGE and Dynamic Light Scattering (DLS) was used. The structure of DAB₃₈₉IL-2 and PEGylated immunotoxin DAB₃₈₉IL-2 was analyzed using the circular dichroism (CD) and fluorescence methods. Also, K562 cells line were treated with various concentrations of DAB₃₈₉IL-2 and conjugated form. In the following, the nuclease activity of DAB₃₈₉IL-2 and PEGylated form was determined. <strong>Results:</strong> The SDS-PAGE result confirmed the site-specific PEGylation of DAB₃₈₉IL-2. Spectroscopy results exhibited that the PEGylation doesn&rsquo;t affect the protein native structure. Also, cytotoxicity assay and nuclease activity test confirmed that PEGylated protein induces death in K562 cells line and DNA degradation respectively. &nbsp;<br> <strong>Conclusion</strong>: It is concluded that the PEGylated immunotoxin DAB₃₈₉IL-2 has a proper structure and function; thus, PEGylated immunotoxin requires more survey due its unique properties.</div> Immunotoxin, PEGylation, DAB389IL-2, Protein Stability, Purification 7 16 http://rmm.mazums.ac.ir/browse.php?a_code=A-10-1110-1&slc_lang=en&sid=1 Nasrin Zarkar 10031947532846008514 10031947532846008514 No Malek Ashtar University of Technology, Tehran, Iran. Mohammad Ali Nasiri Khalili 10031947532846008515 10031947532846008515 No Malek Ashtar University of Technology, Tehran, Iran. Sirus Khodadadi 10031947532846008516 10031947532846008516 Yes Malek Ashtar University of Technology, Tehran, Iran. Mehdi Zeinoddini 10031947532846008517 10031947532846008517 No Malek Ashtar University of Technology, Tehran, Iran. Maryam Ghodrati Siahmazgi 10031947532846008518 10031947532846008518 No Malek Ashtar University of Technology, Tehran, Iran. Nasrin Faramarzi 10031947532846008519 10031947532846008519 No Malek Ashtar University of Technology, Tehran, Iran.