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

• Published in: Iranian journal of science and technology. Transaction A, Science Vol. 43; no. 5; pp. 2091 - 2097
• Main Authors: Bayat, Sh, Zeinoddini, M., Azizi, A., Nasiri Khalili, M. A.
• Format: Journal Article
• Language: English
• Published: Cham Springer International Publishing 01-10-2019
• Subjects: Biology; Chemistry/Food Science; Earth Sciences; Engineering; Life Sciences; Materials Science; Physics; Research Paper; Function; Denileukin diftitox; Immunotoxin; Stability; Expression
• ISSN: 1028-6276; 2364-1819
• DOI: 10.1007/s40995-019-00676-7

Denileukin diftitox
(brand name: Ontak, DAB
389
IL2) is the first recombinant immunotoxin approved by FDA for treatment of CTCL (cutaneous T cell lymphoma). This smart toxin is contains truncated diphtheria toxin (DT
387
), genetically fused to human interleukin 2 (IL2). Like other pharmaceutical productions, there is an aggregation problem in this fusion protein production and achieving a stable protein structure is mainly recommended for clinical use. Herein, we have investigated the effects of co-solvents on the structure and function of
denileukin diftitox
. In this work, the histidine-tagged DAB
389
IL2 was expressed in
Escherichia coli
and after solubilizing and refolding, the inclusion body was purified using nickel chelate affinity chromatography. Then, the structural alterations of the refolded protein in the presence of different co-solvents (sorbitol, trehalose, and sucrose) were investigated using fluorescence and circular dichroism (CD) techniques. Moreover, the nuclease activity of the refolded protein was traced to confirm the refolding process,. Finally, the biological activity of produced DAB
389
IL2 was carried out using MTT assay. The results revealed that, DAB
389
IL2 is more stable in PBS including sorbitol (30 mM) than other stabilizers. The tryptophan residues flexibility—exposure of DAB
389
IL2 to the solvent, in the presence of sorbitol, with respect to fluorescence quenching by acrylamide, indicated the best characterization. Also, in the presence of 2 ng/ml of protease, DAB
389
IL2 was completely undigested and so it indicated the highly stability of the fusion protein to proteases. Furthermore, the refolding and function of the produced fusion protein is verified by DNase activity and bioassay. Therefore, the data of this research could give further insights into the protein refolding mechanism and formulation of the protein in the presence of co-solvents.