Purification and Conformational Characterization of a Novel Interleukin-2 Mutein.
Protein J
; 40(6): 917-928, 2021 12.
Article
em En
| MEDLINE
| ID: mdl-34643845
ABSTRACT
Toxicity of high-dose IL-2-based therapies have motivated the development of the IL-2 mutein, which has low expansion properties for regulatory T lymphocytes. The development of two variants (A and B) for the IL-2 mutein purification as well as a conformational comparative study by Circular dichroism (CD) and fluorescence spectroscopy of these products were evaluated. For the first time, in our center, were used of DTT and 2% SDS in the solubilization step to decrease the aggregates on intermediate product, which favors that disulfide bridges are correctly formed during re-folding. A molecular weight of 18 kDa to the monomeric form and of 25-37 kDa to the oligomeric species were estimated by SDS-PAGE. IL-2 mutein showed similar far-UV CD spectral characteristic typical of cytokines with 41% of α-helix content. Batches obtained by Process B showed similar conformational features according near-UV CD and FS studies. However, those obtained by Process A differed in their folding. IL-2 mutein showed that conformational features by near-UV CD were affected by 2% SDS, no variations on secondary structure were observed. Melting temperature values by far-UV CD were higher than 95 °C, indicating a high thermal stability. Finally, the drug product obtained by Process B showed similar conformational characteristics by near-UV CD and FS, and higher biological activity values (7.0 × 103 ng/mL) in the cell proliferation assay with respect to Process A. Also, the recovery was 15% higher than in the Process A and exhibited a 78.48% of purity. Indeed, Process B was selected for the purification.
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Base de dados:
MEDLINE
Assunto principal:
Interleucina-2
Idioma:
En
Revista:
Protein J
Assunto da revista:
BIOQUIMICA
Ano de publicação:
2021
Tipo de documento:
Article
País de afiliação:
Cuba