Nasrin Zarkar - Malek Ashtar University of Technology, Tehran, Iran.
Mohammad Ali Nasiri Khalili - Malek Ashtar University of Technology, Tehran, Iran.
Sirus Khodadadi - Malek Ashtar University of Technology, Tehran, Iran.
Mehdi Zeinoddini - Malek Ashtar University of Technology, Tehran, Iran.
Maryam Ghodrati Siahmazgi - Malek Ashtar University of Technology, Tehran, Iran.
Nasrin Faramarzi - Malek Ashtar University of Technology, Tehran, Iran.

Background: DAB۳۸۹IL-۲ is considered a fusion immunotoxin and it is used for the CTCL therapy. DAB۳۸۹IL-۲ includes of two distinct portions; the catalytic domain of diphtheria toxin and IL-۲. DAB۳۸۹IL-۲ duo to the presence of a free cysteine residue (Cys ۵۱۳ in IL-۲ 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. Materials and methods: In this study, the PEGylation of recombinant DAB۳۸۹IL-۲ was performed by mPEG-vinylsulfone, through partial denaturation condition at ۴ ۰C for ۲۴ h. To confirm the PEGylation reaction, SDS-PAGE and Dynamic Light Scattering (DLS) was used. The structure of DAB۳۸۹IL-۲ and PEGylated immunotoxin DAB۳۸۹IL-۲ was analyzed using the circular dichroism (CD) and fluorescence methods. Also, K۵۶۲ cells line were treated with various concentrations of DAB۳۸۹IL-۲ and conjugated form. In the following, the nuclease activity of DAB۳۸۹IL-۲ and PEGylated form was determined. Results: The SDS-PAGE result confirmed the site-specific PEGylation of DAB۳۸۹IL-۲. Spectroscopy results exhibited that the PEGylation doesn’t affect the protein native structure. Also, cytotoxicity assay and nuclease activity test confirmed that PEGylated protein induces death in K۵۶۲ cells line and DNA degradation respectively.   Conclusion: It is concluded that the PEGylated immunotoxin DAB۳۸۹IL-۲ has a proper structure and function; thus, PEGylated immunotoxin requires more survey due its unique properties.

Immunotoxin, PEGylation, DAB۳۸۹IL-۲, Protein Stability, Purification