Bifunctional GM-CSF-derived peptides as tools for O-glycoengineering and protein tagging
Autor: | Natalia Ceaglio, María de los Milagros Sales, Ricardo Kratje, Marcos Oggero |
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Rok vydání: | 2021 |
Předmět: |
0106 biological sciences
0301 basic medicine Macrophage colony-stimulating factor Glycosylation medicine.drug_class Bioengineering Peptide Protein Engineering Monoclonal antibody Antiviral Agents 01 natural sciences Applied Microbiology and Biotechnology Epitope 03 medical and health sciences chemistry.chemical_compound 010608 biotechnology medicine Animals chemistry.chemical_classification Antibodies Monoclonal Granulocyte-Macrophage Colony-Stimulating Factor Biological activity General Medicine Fusion protein Recombinant Proteins In vitro Rats Sialic acid 030104 developmental biology chemistry Biochemistry Peptides Biotechnology |
Zdroj: | Journal of Biotechnology. 327:18-27 |
ISSN: | 0168-1656 |
Popis: | Rapid development of effective biotherapeutics has been a concern during the last couple decades. In our work we designed two novel peptide tags, GMOP and mGMOP, derived from the N-terminal region of human granulocyte and macrophage colony stimulating factor (hGM-CSF), which contain four and six potential O-glycosylation sites, respectively. These peptide tags were fused to the N-terminus of human interferon-α2b (hIFN-α2b), a therapeutic antiviral and antiproliferative protein rapidly cleared from circulation. Two new molecules were obtained which, consistently with the presence of O-glycans, showed higher molecular masses, more negatively charged isoforms, and higher sialic acid content compared to wild-type IFN. In vitro bioactivity of purified chimeras revealed a similar antiviral specific biological activity (SBA) compared to unmodified IFN. A reduction of antiproliferative SBA was only observed for mGMOP-IFN. Pharmacokinetic studies in rats showed a notable improvement in terminal half-life (t1/2elim) (3.3 and 2.8 times-longer) and a marked reduction of the apparent clearance (CLapp, 3.7 and 4.1-fold lower for GMOP-IFN and mGMOP-IFN in comparison with native IFN, respectively). Furthermore, the in vitro thermal and plasma stability of both proteins was improved. Finally, a monoclonal antibody (mAb) that recognizes an N-terminal GM-CSF epitope was able to bind both chimeras in western blots and ELISAs. This demonstrates the potential of both peptides to behave as bifunctional tags to create novel long-acting biotherapeutics and to facilitate detection and purification. |
Databáze: | OpenAIRE |
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