Volume 8, Issue 2 (May 2020)                   Res Mol Med (RMM) 2020, 8(2): 83-92 | Back to browse issues page


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Abstract:   (2091 Views)
Background: Denileukin diftitox (trade name, Ontak) is the first recombinant immunotoxin (IM), in which the binding domain of diphtheria toxin has been replaced by the amino acid sequence of human interleukin-2 (DT389IL-2) using genetic engineering. Purity, stability, and structural property of the protein are critical factors for the scale-up production of this fusion protein. In this IM, location 519 has free cysteine residue that leads to cross S-S bound formation in the refolding process and, as a result, misfolding/aggregation of the protein may occur.
Materials and Methods: To inhibit misfolding/aggregation, we substituted cysteine 519 by a serine residue with site-directed mutagenesis, and then the ability of the mutated protein for binding to the IL-2 receptor was predicted and determined by bioinformatics tools. For this purpose, the sequence of the denileukin diftitox was adopted from Drugbank, and the mentioned substitution applied. Two methods determined the folding of the fusion protein: de novo modeling method (by utilizing the I-TASSER database) and homology modeling method (by using some databases and tools, including Swiss-Model, PHYRE2, M4T, ModWeb, RaptorX, and EasyModeller). Finally, the ability of the proteins for binding to the IL-2 receptor was investigated by pyDock and Zdock docking servers, as well as Hex software.
Results: The result showed that the mutated form (C519S) of this protein folds appropriately, and the ΔG of the models, measured by STRUM, showed no significant variation.  Also, docking analysis has shown that the protein can efficiently bind to the IL-2 receptor without any substantial changes in the binding energy.
Conclusion: The present study shows that the suggested mutation of this protein can be an acceptable replacement for denileukin diftitox with a similar affinity and a more proper refolding process.
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Type of Study: Research | Subject: Biology
Received: 2020/01/2 | Accepted: 2020/04/14 | Published: 2020/05/21