Engineering of recombinant Staphylococcal protein A with cysteine residue (SPA-Cys) for preparation of affi nity chromatography stationary phase and formation of bioselective element of immunosensor. Methods. DNA sequences encoding IgG-binding region of SPA, His-tag and cysteine were genetically fused and expressed in E. coli. SPA-Cys was immobilized on maleimide-functionalized silica beads for affi nity chromatography stationary phase preparation and on a gold sensor surface as a bioselective element of immunosensor. Results. SPA-Cys was expressed at a high-level in a soluble form. The target protein was purifi ed and showed a high IgG-binding activity. The capacity of the obtained SPA-Cys-based affi nity chromatography stationary phase was 10-12 mg of IgG /ml. The purity of eluted IgG was more than 95 % in one-step purifi cation procedure. The developed SPA-Cys-based bioselective element of immunosensor selectively interacted with human IgG and did not interact with the control proteins. Conclusions. The recombinant Staphylococcal protein A with cysteine residue was successfully used for the preparation of affi nity chromatography stationary phase and formation of the bioselective element of immunosensor. K e y w o r d s: antibodies, recombinant Staphylococcal protein A, protein immobilization, affi nity chromatography, immunosensor, surface plasmon resonance.
To investigate the formation of an intermediate layer of the immunosensor bioselective element based on the recombinant protein A from Staphylococcus aureus with cysteine residue (SPA-Cys) and its interactions with human IgG using the SPR spectrometer «Plasmon». Methods. The activity of the immune components applied was tested by ELISA. The spectrometry of surface plasmon resonance was used for studying protein immobilization on a gold sensor surface and interactions between the immobilized SPA-Cys and human immunoglobulin. Results. A direct dependence of the sensor response on the concentration of SPA-Cys in the range of 0.2 to 2 μM at its immobilization was demonstrated. The efficiency of blocking nonspecifi c adsorption sites on the sensor surface with milk proteins and the direct dependence of the sensor response on IgG concentration and surface density of immobilized SPA-Cys were shown. Fitting the experimental data to a Langmuir plot yields a K d value for SPA-Cys/IgG binding 8.5 ± 0.7× 10 -8 M (K a = 1.2 ± 0.1× 10 7 M -1 ). The determined equilibrium binding constant indicates a quite strong interaction and its value is consistent with the literature data. Conclusions. A successful immobilization of SPA-Cys on a gold surface of the SPR spectrometer while preserving its high immunoglobulin-binding activity, selectivity and stability of the sensor response confi rms the effi ciency of SPA-Cys as an intermediate component for the creation of the immunosensor bioselective elements. K e y w o r d s: immunoglobulin, recombinant Staphylococcal protein A, surface plasmon resonance, protein immobilization, immunosensor, equilibrium binding constant.
The developed surface plasmon resonance (SPR) biosensor based on the recombinant Staphylococcal protein A with an additional cysteine residue (SPA-Cys) used as a biorecognition component showed a good selectivity and sensitivity for the immunoglobulin detection. The developed biosensor with SPA-Cys-based bioselective element can also be used as a first step of immunosensor creation. The successful immobilization of SPA-Cys on the nanolayer gold sensor surface of the SPR spectrometer was performed. The efficiency of blocking nonspecific sorption sites on the sensor surface with milk proteins, gelatin, BSA, and HSA was studied, and a rather high efficiency of using gelatin was confirmed. The SPR biosensor selectively interacted with IgG and did not interact with the control proteins. The linear dependence of the sensor response on the IgG concentration in the range from 2 to 10 μg/ml was shown. Using the calibration curve, the IgG concentration was measured in the model samples. The determined concentrations are in good agreement (r
2 = 0.97) with the given concentration of IgG.
Comparison of the IgG-binding activity of recombinant Staphylococcal protein A with introduced C-terminal cysteine residue (SPA-Cys) or goat anti-human IgG antibodies (anti-IgG) after their immobilization on a gold sensor surface of surface plasmon resonance (SPR) spectrometer. Methods. SPA-Cys or anti-IgG were immobilized on a gold sensor surface to form two variants of a bioselective element of the immunosensor. SPR spectrometry was used for the detection of IgG-binding activity of the immobilized proteins. Results. The SPR sensor response to the immobilization of anti-IgG was more than two times higher than that to the immobilization of SPA-Cys. However, there is almost the double advantage for SPA-Cys in the number of immobilized molecules. Moreover, the bioselective element of the immunosensor based on SPA-Cys showed a much better capability of binding IgG than the bioselective element based on anti-IgG. Conclusions. Comparison of immobilization of SPA-Cys or anti-IgG on the sensor surface of SPR spectrometer, and the interactions of immobilized proteins with human IgG demonstrated obvious advantages of SPA-Cys.
Comparison of IgG-binding activity of Staphylococcal protein A (SPA) and recombinant SPA with specially introduced C-terminal cysteine residue (SPA-Cys) after their immobilization on a gold sensor surface of the surface plasmon resonance (SPR) spectrometer. Methods. SPA or SPA-Cys was immobilized on a gold sensor surface to form two variants of bioselective elements of biosensor. SPR spectrometry was used for detection of IgG-binding activity of the immobilized proteins. Results. The SPR sensor response to the immobilization of SPA was more than three times less than that to immobilization of SPA-Cys. SPA-Cys demonstrates also almost 4-fold advantage in the number of immobilized molecules. Moreover, the bioselective element based on SPA-Cys showed a much better capability of binding IgG than the bioselective element based on SPA. Conclusions. The study of the processes of immobilization of SPA or SPA-Cys on the sensor surface of SPR spectrometer, and the interactions of immobilized proteins with human IgG demonstrated obvious advantages of recombinant protein A.
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