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- Title
Expression, Purification, and Biological Evaluation of XTEN-GCSF in a Neutropenic Rat Model.
- Authors
Nikravesh, Fatemeh Yadavar; Gholami, Parisa; Bayat, Elham; Talebkhan, Yeganeh; Mirabzadeh, Esmat; Damough, Shadi; Aliabadi, Hooman Aghamirza Moghim; Nematollahi, Leila; Ardakani, Yalda Hosseinzadeh
- Abstract
Granulocyte colony-stimulating factor (GCSF) stimulates the proliferation of neutrophils but it has low serum half-life. Therefore, the present study was done to investigate the effect of XTENylation on biological activity, pharmacokinetics, and pharmacodynamics of GCSF in a neutropenic rat model. XTEN tag was genetically fused to the N-terminal region of GCSF-encoding gene fragment and subcloned into pET28a expression vector. The cytoplasmic expressed recombinant protein was characterized through intrinsic fluorescence spectroscopy (IFS), dynamic light scattering (DLS), and size exclusion chromatography (SEC). In vitro biological activity of the XTEN-GCSF protein was evaluated on NFS60 cell line. Hematopoietic properties and pharmacokinetics were also investigated in a neutropenic rat model. An approximately 140 kDa recombinant protein was detected on SDS-PAGE. Dynamic light scattering and size exclusion chromatography confirmed the increase in hydrodynamic diameter of GCSF molecule after XTENylation. GCSF derivatives showed efficacy in proliferation of NFS60 cell line among which the XTEN-GCSF represented the lowest EC50 value (100.6 pg/ml). Pharmacokinetic studies on neutropenic rats revealed that XTEN polymer could significantly increase protein serum half-life in comparison with the commercially available GCSF molecules. PEGylated and XTENylated GCSF proteins were more effective in stimulation of neutrophils compared to the GCSF molecule alone. XTENylation of GCSF represented promising results in in vitro and in vivo studies. This approach can be a potential alternative to PEGylation strategies for increasing serum half-life of protein.
- Subjects
GENE expression; ANIMAL disease models; GRANULOCYTE-colony stimulating factor; GEL permeation chromatography; BLOOD proteins; POLYMERS
- Publication
Applied Biochemistry & Biotechnology, 2024, Vol 196, Issue 2, p804
- ISSN
0273-2289
- Publication type
Article
- DOI
10.1007/s12010-023-04522-w