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Expression, Purification, and Biological Evaluation of XTEN-GCSF in a Neutropenic Rat Model

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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.

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Acknowledgements

The authors wish to express their deep gratitude to all who provided deep technical supports.

Funding

This project was supported by Pasteur Institute of Iran and the Biotechnology Development Headquarter of the Vice-Presidency for Science & Technology, Tehran, Iran.

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Authors and Affiliations

  1. Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran

    Fatemeh Yadavar Nikravesh, Parisa Gholami, Elham Bayat, Yeganeh Talebkhan, Shadi Damough, Hooman Aghamirza Moghim Aliabadi & Leila Nematollahi

  2. Department of Molecular Medicine, Pasteur Institute of Iran, Tehran, Iran

    Esmat Mirabzadeh

  3. Department of Pharmaceutics, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran

    Yalda Hosseinzadeh Ardakani

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  1. Fatemeh Yadavar Nikravesh
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Contributions

YT, LN, and YHA: Design the study and revise of the manuscript. FYN: Major experimental work, data analysis, and contribution in writing the manuscript. PGh, EB, ShD, and HAMA: Contribution in experimental work. EM: Contribution in animal studies.

Corresponding authors

Correspondence to Yeganeh Talebkhan or Yalda Hosseinzadeh Ardakani.

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All experiments and procedures were approved by the Ethics Committee of Pasteur Institute of Iran (IR.PII.REC.1399.013) and performed in accordance with the approved guidelines and regulations.

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Fig. S1

Western blotting using HRP conjugated rabbit anti-His antibody. #1, 2: Recombinant E. coli cell lysates before and after induction; #3, 4: Purified XTEN-GCSF samples; M: Pre-stained protein molecular weight marker. (PNG 88 kb)

High resolution image (TIF 153 kb)

Fig. S2

Dynamic light scattering histogram. Assessment of the size of GCSF derivatives. (PNG 760 kb)

Fig. S3

Blood counting of rats received single doses of GCSF derivatives. (a) Filgrastim; (b) PEG-Filgrastim; (c) XTEN-GCSF. Data are means ± SE SD of 3 random rats/group on a linear scale. Bars represent the SD values. (PNG 17 kb)

(PNG 19 kb)

High resolution image (TIFF 160 kb)

High resolution image (TIFF 170 kb)

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Nikravesh, F.Y., Gholami, P., Bayat, E. et al. Expression, Purification, and Biological Evaluation of XTEN-GCSF in a Neutropenic Rat Model. Appl Biochem Biotechnol 196, 804–820 (2024). https://doi.org/10.1007/s12010-023-04522-w

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