Modelling of immunosensor response: the evaluation of binding kinetics between an immobilized receptor and structurally-different genetically engineered ligands

Abstract

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

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

“…The binding kinetics assay of the purified heterodimers and monomeric SCF to a surface-immobilized G-CSF and SCF receptors was performed by total internal reflection ellipsometry (TIRE). The binding assays of monomeric G-CSF and SCF-Lα-GCSF to GCSF-R were described in Balevicius et al (2019) . GCSF-R and c-kit used for TIRE are chimeric proteins (both obtained from Abcam).…”

“…The reaction chamber with a volume of 0.028 mL was positioned underneath the cell layer of the slide. Two gold sensor disks for immobilization of GCSF-R and c-kit were prepared as described previously ( Balevicius et al, 2014 , 2019 ) with slight modifications. Briefly, the BK7-glass/Cr-Au slides were cleaned using the BAL-TEC SCD 050 Sputter Coater plasma cleaner, washed with methanol for 10 min, and then rinsed with hexane for 5 min.…”

“…Full-size  DOI: 10.7717/peerj.9788/ fig-3 and GCSF-R were 10-fold lower than that of monomeric G-CSF (Balevicius et al, 2019).…”

“…A. Woollam Co., Inc. data, Lincoln, NE, USA). The binding kinetics of heterodimers and monomeric SCF were evaluated by a standard fully reversible Langmuir kinetics model described previously (Balevicius et al, 2019).…”

“…In this study, we described the development of two heterodimeric fusion proteins composed of human SCF and human G-CSF connected via a peptide linker, which ensured the activity of two monomers in the heterodimeric molecule (Arai et al, 2001;Mickiene et al, 2017;Balevicius et al, 2019). The heterodimers were purified, characterized, and compared with monomeric human G-CSF and SCF.…”