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In this research we have evaluated the binding kinetics between an immobilized receptor and several genetically engineered ligands, differing by molecular mass or by the number of binding sites available for the binding to the receptor. Genetically engineered protein (GCSF-Receptor), which contains some antibody parts (Fc domain) and at some extent is similar to antibody because also has two binding sites that selectively bind another proteinglycoprotein granulocyte colony stimulating factor (GCSF), which was immobilized on a thin gold layer in order to design an immunosensor sensitive to GCSF. Three structurally different GCSF-based proteins were genetically-engineered and evaluated as ligands, which selectively bind to immobilized GCSF-Receptor: (i) GCSF monomer (mGCSF), (ii) GCSF-homodimer consisting of two via polypeptide Lα-based linker 'fused' GCSF molecules ((GCSF)2Lα) and (iii) GCSFheterodimer (SCF-Lα-GCSF), which is based on a native GCSF molecule 'fused' via Lα-based linker with another proteina soluble part of stem cell factor (SCF). SCF, unlike GCSF, does not contain any site suitable for GCSF-Receptor binding. The ligands differ by: (i) molecular mass -(GCSF)2Lα and SCF-Lα-GCSF F are two times heavier than mGCS, (ii) number of binding sites -mGCSF and SCF-Lα-GCSF have one binding site, while (GCSF)2Lα has two. The binding kinetics of mGCSF, (GCSF)2Lα, and SCF-Lα-GCSF with immobilized GCSF-Receptor was investigated using total internal reflection ellipsometry. The interaction kinetics of the mGCSF and SCF-Lα-GCSF are both well described using a standard Langmuir kinetics model. However, receptor-ligand association and dissociation rates in the case of SCF-Lα-GCSF ligand are about 10 times lower than that of mGCSF. The association rate of (GCSF)2Lα

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Granulocyte colony-stimulating factor (G-CSF) has found widespread clinical application, and modified forms with improved biopharmaceutical properties have been marketed as well. PEGylation, the covalent modification of G-CSF with polyethylene glycol (PEG), has a beneficial effect on drug properties, but there are concerns connected to the immunogenicity of PEGylated compounds and bioaccumulation of the synthetic polymer. To overcome challenges connected with chemical modifications, we developed fusion proteins composed of two G-CSF molecules connected via different peptide linkers. Three different homodimeric G-CSF proteins were purified, and their in vitro and in vivo activities were determined. A G-CSF dimer, GCSF-Lα, was constructed using an alpha-helix-forming peptide linker, and it demonstrated an extended half-life in serum with a stronger neutrophil response as compared to the monomeric G-CSF protein. The GCSF-Lα protein, therefore, might be selected for further studies as a potential drug candidate.

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