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Mickiene G, Dalgėdienė I, Zvirblis G, et al. Human granulocyte-colony stimulating factor (G-CSF)/stem cell factor (SCF) fusion proteins: design, characterization and activity. PeerJ. 2020;8:e9788doi: 10.7717/peerj.9788.
Mickiene, G., Dalgėdienė, I., Zvirblis, G., Dapkunas, Z., Plikusiene, I., Buzavaite-Verteliene, E., Balevičius, Z., Rukšėnaitė, A., & Pleckaityte, M. (2020). Human granulocyte-colony stimulating factor (G-CSF)/stem cell factor (SCF) fusion proteins: design, characterization and activity. PeerJ, 8e9788. https://doi.org/10.7717/peerj.9788
Mickiene, Gitana, et al. "Human granulocyte-colony stimulating factor (G-CSF)/stem cell factor (SCF) fusion proteins: design, characterization and activity." PeerJ vol. 8 (2020): e9788. doi: https://doi.org/10.7717/peerj.9788
Mickiene G, Dalgėdienė I, Zvirblis G, Dapkunas Z, Plikusiene I, Buzavaite-Verteliene E, Balevičius Z, Rukšėnaitė A, Pleckaityte M. Human granulocyte-colony stimulating factor (G-CSF)/stem cell factor (SCF) fusion proteins: design, characterization and activity. PeerJ. 2020 Aug 21;8:e9788. doi: 10.7717/peerj.9788. eCollection 2020. PMID: 32884863; PMCID: PMC7444511.
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PeerJ. 2020 Aug 21;8:e9788. doi: 10.7717/peerj.9788. eCollection 2020.

Human granulocyte-colony stimulating factor (G-CSF)/stem cell factor (SCF) fusion proteins: design, characterization and activity.

PeerJ

Gitana Mickiene, Indrė Dalgėdienė, Gintautas Zvirblis, Zilvinas Dapkunas, Ieva Plikusiene, Ernesta Buzavaite-Verteliene, Zigmas Balevičius, Audronė Rukšėnaitė, Milda Pleckaityte

Affiliations

  1. Institute of Biotechnology, Vilnius University, Vilnius, Lithuania.
  2. Profarma UAB, Vilnius, Lithuania.
  3. Department of Physical Chemistry, Faculty of Chemistry and Geosciences, Vilnius University, Vilnius, Lithuania.
  4. Plasmonics and Nanophotonics Laboratory, Department of Laser Technology, Center for Physical Sciences and Technology, Vilnius, Lithuania.

PMID: 32884863 PMCID: PMC7444511 DOI: 10.7717/peerj.9788

Abstract

BACKGROUND: Stem cell factor (SCF) and granulocyte-colony stimulating factor (G-CSF) are well-characterized vital hematopoietic growth factors that regulate hematopoiesis. G-CSF and SCF synergistically exhibit a stimulatory effect on hematopoietic progenitors. The combination of G-CSF and SCF has been used for mobilization of peripheral blood progenitor cells in cancer and non-cancerous conditions. To overcome challenges connected with the administration of two cytokines, we developed two fusion proteins composed of human SCF and human G-CSF interspaced by an alpha-helix-forming peptide linker.

METHODS: The recombinant proteins SCF-Lα-GCSF and GCSF-Lα-SCF were purified in three steps using an ion-exchange and mixed-mode chromatography. The purity and quantity of the proteins after each stage of purification was assessed using RP-HPLC, SDS-PAGE, and the Bradford assays. Purified proteins were identified using high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) and the Western blot analyses. The molecular weight was determined by size exclusion HPLC (SE-HPLC). The activity of heterodimers was assessed using cell proliferation assays in vitro. The capacity of recombinant fusion proteins to stimulate the increase of the absolute neutrophil count in rats was determined in vivo. The binding kinetics of the proteins to immobilized G-CSF and SCF receptors was measured using total internal reflection ellipsometry and evaluated by a standard Langmuir kinetics model.

RESULTS: The novel SCF-Lα-GCSF and GCSF-Lα-SCF proteins were synthesized in

© 2020 Mickiene et al.

Keywords: Absolute neutrophil count; Activity; Cytokines; Fusion proteins; G-CSF; Hematopoiesis; Linker; Protein purification; SCF

Conflict of interest statement

Gitana Mickiene and Zilvinas Dapkunas are employes of UAB Profarma, Vilnius.

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