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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 Escherichia coli . The purity of the heterodimers reached >90% as determined by RP-HPLC. The identity of the proteins was confirmed using the Western blot and HPLC/ESI-MS assays. An array of multimeric forms, non-covalently associated dimers or trimers were detected in the protein preparations by SE-HPLC. Each protein induced a dose-dependent proliferative response on the cell lines. At equimolar concentration, the heterodimers retain 70-140% of the SCF monomer activity ( p ≤ 0.01) in promoting the M-07e cells proliferation. The G-CSF moiety in GCSF-Lα-SCF retained 15% ( p ≤ 0.0001) and in SCF-Lα-GCSF retained 34% ( p ≤ 0.01) of the monomeric G-CSF activity in stimulating the growth of G-NFS-60 cells. The obtained results were in good agreement with the binding data of each moiety in the fusion proteins to their respective receptors. The increase in the absolute neutrophil count in rats caused by the SCF-Lα-GCSF protein corresponded to the increase induced by a mixture of SCF and G-CSF., Competing Interests: Gitana Mickiene and Zilvinas Dapkunas are employes of UAB Profarma, Vilnius., (© 2020 Mickiene et al.)

The modelling of protein-protein binding kinetics is important for the development of affinity-sensors and the prediction of signaling protein based drug efficiency. Therefore, in this research we have evaluated the binding kinetics of several genetically designed protein models: (i) three different ligands based on granulocyte colony-stimulating factor GCSF homo-dimeric derivatives linked by differed by linkers of different length and flexibility; (ii) an antibody-like receptor (GCSF-R) based on two GCSF-receptor sites immobilized to Fc domains, which are common parts of protein structures forming antibodies. Genetically engineered GCSF-R is similar to an antibody because it, like the antibody, has two binding sites, which both selectively bind with GCSF ligands. To design the affinity sensor model studied here, GCSF-R was immobilized on a thin gold layer via self-assembled monolayer conjugated with Protein-G. Binding kinetics between immobilized GCSF-R and all three different recombinant GCSF-based homo-dimeric derivatives were evaluated by total internal reflection ellipsometry. Association constants were determined by fitting mathematical models to the experimental data. It was clearly observed that both (i) affinity and (ii) binding kinetics depend on the length and flexibility of the linker that connects both domains of a GCSF-based ligand. The fastest association between immobilized GCSF-R and GCSF-based ligands was observed for ligands whose GCSF domains were interconnected by the longest and the most flexible linker. Here we present ellipsometry-based measurements and models of the interaction kinetics that advance the understanding of bidentate-receptor-based immunosensor action and enables us to predict the optimal linker structure for the design of GCSF-based medications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)