Co-Solvents Effects on the Stability of Recombinant Immunotoxin Denileukin Diftitox: Structure and Function Assessment

Bayat, Sh.; Zeinoddini, Mehdi; Azizi, Azadeh; Khalili, Mohammad Ali Nasiri

doi:10.1007/s40995-019-00676-7

Cited by 7 publications

(5 citation statements)

References 26 publications

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“…DAB389IL-2 is a recombinant immunotoxin composed of a catalytic moiety of diphtheria toxin fused to the binding moiety of IL-2 [20]. In the purification prosses of Ontak, many problems are showing up, including misfolding and instability [21]. In the present study, to solve these problems, the molecular and structural studies were exploited.…”

Section: Discussionmentioning

confidence: 99%

Molecular Docking and In Silico Study of Denileukin Diftitox: Comparison of Wild Type With C519S Mutant

et al. 2020

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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(trade name, Ontak) is the first recombinant immunotoxin (IM), in which the binding domain of diphtheria toxin has been replaced by the amino acid sequence of human interleukin-2 (DT389IL-2) using genetic engineering.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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Section: Discussionmentioning

confidence: 99%

Molecular Docking and In Silico Study of Denileukin Diftitox: Comparison of Wild Type With C519S Mutant

et al. 2020

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“…A variety of structures of ITs were constructed, composed of a truncated diphtheria toxin (residues 1-389; DT389) (Expasy accession no. P00588) [49] and different Yi-Fan Zhang's humanized anti-GPC3 scFvs (YP7, YP8, YP9 and YP9.1) [28], using credible in-silico software. This preliminary phase of studies focused on investigating the effectiveness of different G 4 S linkers between toxin and scFv moieties on secondary and three-dimensional structures, stability, flexibility, solubility, and physic-chemical properties.…”

Section: Construction Designmentioning

confidence: 99%

DT389-YP7, a Recombinant Immunotoxin against Glypican-3 That Inhibits Hepatocellular Cancer Cells: An In Vitro Study

Yeganeh

Heiat

Kieliszek

et al. 2021

Toxins

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Hepatocellular carcinoma (HCC) is one of the high-metastatic types of cancer, and metastasis occurs in one-third of patients with HCC. To maintain the effectiveness of drug compounds on cancer cells and minimize their side effects on normal cells, it is important to use new approaches for overcoming malignancies. Immunotoxins (ITs), an example of such a new approach, are protein-structured compounds consisting of toxic and binding moieties which can specifically bind to cancer cells and efficiently induce cell death. Here, we design and scrutinize a novel immunotoxin against an oncofetal marker on HCC cells. We applied a truncated diphtheria toxin (DT389) without binding domain as a toxin moiety to be fused with a humanized YP7 scFv against a high-expressed Glypican-3 (GPC3) antigen on the surface of HCC cells. Cytotoxic effects of this IT were investigated on HepG2 (GPC3+) and SkBr3 (GPC3−) cell lines as positive- and negative-expressed GPC3 antigens. The dissociation constant (Kd) was calculated 11.39 nM and 18.02 nM for IT and YP7 scfv, respectively, whereas only IT showed toxic effects on the HepG2 cell line, and decreased cell viability (IC50 = 848.2 ng/mL). Changing morphology (up to 85%), cell cycle arrest at G2 phase (up to 13%), increasing intracellular reactive oxygen species (ROSs) (up to 50%), inducing apoptosis (up to 38% for apoptosis and 23% for necrosis), and an almost complete inhibition of cell movement were other effects of immunotoxin treatment on HepG2 cells, not on SkBr3 cell line. These promising results reveal that this new recombinant immunotoxin can be considered as an option as an HCC inhibitor. However, more extensive studies are needed to accomplish this concept.

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“…In this sequence, the NdeI and EcoRI restriction sites were added into the 5́and 3΄ ends of the corresponding gene, respectively. Also, six-histidine codons (CAC) 6 , was added after the start codon (ATG) at the N-terminus to facilitate the process of purification [34,35]. The recombinant plasmid pET21a Applied Biochemistry containing the DAB 389 IL-2 gene was transferred to the E. coli strain Rosetta-gami (DE3) competent cells.…”

Section: Dab 389 Il-2 Expressionmentioning

confidence: 99%

Expression and purification of soluble and functional fusion protein DAB₃₈₉IL‐2 into the E. coli strain Rosetta‐gami (DE3)

Zarkar

Khalili

Khodadadi

et al. 2019

Biotech and App Biochem

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DAB389IL‐2 (Denileukin diftitox) is considered an immunotoxin, and it is the first immunotoxin approved by Food and Drug Administration. It is used for the treatment of a cutaneous form of T‐cell lymphoma. This fusion protein has two disulfide bonds in its structure that play an essential role in toxicity and functionality of the immunotoxin. Escherichia coli (E. coli) strain BL21 (DE3) is not capable of making disulfide bonds in its reductive cytoplasm, but the E. coli strain Rosetta‐gami (DE3) is a proper strain for the correct expression of the protein due to mutations in glutaredoxin reductase and thioredoxin reductase. In this study, a pET21a vector with the His6‐tag fused at the N‐terminus of DAB389IL‐2 was used to express the soluble immunotoxin in E. coli Rosetta‐gami (DE3). After the purification of the soluble protein by two‐step column chromatographies, the structure of DAB389IL‐2 was analyzed using the Native‐PAGE and circular dichroism methods. In the following, the nuclease activity of soluble DAB389IL‐2 and its cytotoxicity activity were determined. It is concluded that the soluble recombinant protein expressed in the E. coli Rosetta‐gami (DE3) has an intact structure and also functional; hence, this form of immunotoxin could be competitive with its commercial counterparts.

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Co-Solvents Effects on the Stability of Recombinant Immunotoxin Denileukin Diftitox: Structure and Function Assessment

Cited by 7 publications

References 26 publications

Molecular Docking and In Silico Study of Denileukin Diftitox: Comparison of Wild Type With C519S Mutant

Molecular Docking and In Silico Study of Denileukin Diftitox: Comparison of Wild Type With C519S Mutant

DT389-YP7, a Recombinant Immunotoxin against Glypican-3 That Inhibits Hepatocellular Cancer Cells: An In Vitro Study

Expression and purification of soluble and functional fusion protein DAB₃₈₉IL‐2 into the E. coli strain Rosetta‐gami (DE3)

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Co-Solvents Effects on the Stability of Recombinant Immunotoxin Denileukin Diftitox: Structure and Function Assessment

Cited by 7 publications

References 26 publications

Molecular Docking and In Silico Study of Denileukin Diftitox: Comparison of Wild Type With C519S Mutant

Molecular Docking and In Silico Study of Denileukin Diftitox: Comparison of Wild Type With C519S Mutant

DT389-YP7, a Recombinant Immunotoxin against Glypican-3 That Inhibits Hepatocellular Cancer Cells: An In Vitro Study

Expression and purification of soluble and functional fusion protein DAB389IL‐2 into the E. coli strain Rosetta‐gami (DE3)

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Product

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Expression and purification of soluble and functional fusion protein DAB₃₈₉IL‐2 into the E. coli strain Rosetta‐gami (DE3)