Journal
BIOMOLECULES
Volume 12, Issue 8, Pages -Publisher
MDPI
DOI: 10.3390/biom12081111
Keywords
biotherapeutics; cytotoxin; diphtheria toxin; IL-13; inclusion bodies; refolding; disulfide bond; protein oligomerization; MALS; SAXS; LC/MS
Categories
Funding
- European Union from the European Regional Development Fund under the Smart Growth Operational Program 2014-2020
- National Center for Research and Development [POIR.01.01.01-00-0912/17-00, 5/1.1.1/2017]
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Protein aggregation is a common problem in industrial preparation and understanding its structure and function is crucial for the development of therapeutic proteins. This study analyzed the side products of a single-chain fusion cytotoxin and provided comprehensive insights into their molecular architecture and function.
Protein aggregation is a well-recognized problem in industrial preparation, including biotherapeutics. These low-energy states constantly compete with a native-like conformation, which is more pronounced in the case of macromolecules of low stability in the solution. A better understanding of the structure and function of such aggregates is generally required for the more rational development of therapeutic proteins, including single-chain fusion cytotoxins to target specific receptors on cancer cells. Here, we identified and purified such particles as side products of the renaturation process of the single-chain fusion cytotoxin, composed of two diphtheria toxin (DT) domains and interleukin 13 (IL-13), and applied various experimental techniques to comprehensively understand their molecular architecture and function. Importantly, we distinguished soluble purified dimeric and fractionated oligomeric particles from aggregates. The oligomers are polydisperse and multimodal, with a distribution favoring lower and even stoichiometries, suggesting they are composed of dimeric building units. Importantly, all these oligomeric particles and the monomer are cystine-dependent as their innate disulfide bonds have structural and functional roles. Their reduction triggers aggregation. Presumably the dimer and lower oligomers represent the metastable state, retaining the native disulfide bond. Although significantly reduced in contrast to the monomer, they preserve some fraction of bioactivity, manifested by their IL-13RA2 receptor affinity and selective cytotoxic potency towards the U-251 glioblastoma cell line. These molecular assemblies probably preserve structural integrity and native-like fold, at least to some extent. As our study demonstrated, the dimeric and oligomeric cytotoxin may be an exciting model protein, introducing a new understanding of its monomeric counterpart's molecular characteristics.
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