OPTICAL BIOSENSOR AS A TOOL FOR CHARACTERIZATION OF HUMAN IFN-GAMMA - RECEPTOR INTERACTION
ELENA KRACHMAROVA1*, DAMIEN MAURIN2, HUGUES LORTAT-JACOB2 AND GENOVEVA NACHEVA1
1 – Institute of Molecular Biology “Roumen Tsanev”, Bulgarian Academy of Sciences, Sofia, Bulgaria
2 – Institut de Biologie Structurale, Grenoble, France
*Corresponding author: elenakrachmarova@bio21.bas.bg
Keywords: surface plasmon resonance, human interferon-gamma, human interferon-gamma receptor, thermodynamic parameters
Abstract: In the recent years, optical biosensors, such as Biacore™ system, have become increasingly popular for studying macromolecular interactions. The surface plasmon resonance (SPR) underlying the Biacore™ systems is a rapid method for analysing interactions between different molecules without the need for their pre-labelling. Biacore™ is based on a sensor chip with biospecific surface on which the ligand-analyte interaction takes place. Binding of molecules to the sensor surface generates a response, which is proportional to the bound mass, and can be detected down to changes of a few picograms per square millimetre, corresponding to concentrations of picomolar to nanomolar range in the bulk sample solution. An optical system is responsible for generating and reporting the SPR signal. The data generated by Biacore™ evaluation software gives information about the specificity of the binding, kinetics (rate of the association/disassociation) and affinity (strength) of the binding.
Human interferon-gamma (hIFNγ) exerts a wide range of immunoregulatory activities. The therapeutic applications of this cytokine challenged us to develop and optimize a highly efficient technology for its soluble production and purification. It is based on the fusion with solubility-enhancing SUMO-protein and expression in chaperon strain of E. coli BL21(DE3)/pG-KJE8. By using this methodology, we obtained highly pure and biologically active hIFNγ. SPR was used to evaluate its binding affinity to the extracellular part of the hIFNγ receptor (hIFNGR1). The resulting sensograms showed high affinity and establishment of strong hIFNγ/hIFNGR1 complex regardless of the analysed concentration of hIFNγ. These data are another confirmation for the effectiveness of the developed by us SUMO-methodology, since it yields recombinant protein with preserved biological characteristic. The obtained thermodynamic parameters will be used for development and validation of in silico model of the hIFNγ/IFNGR1 interaction and further identification of the structural motifs important for the formation of stable complex.