Papers by Anna Bakhmachuk
Biopolymers and Cell, 2016
Comparison of the IgG-binding activity of recombinant Staphylococcal protein A with introduced C-... more 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.
Biopolymers and Cell, 2015
To investigate the formation of an intermediate layer of the immunosensor bioselective element ba... more 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.
Biopolymers and Cell, 2015
Engineering of recombinant Staphylococcal protein A with cysteine residue (SPA-Cys) for preparati... more 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.
Aim. Comparison of the IgG-binding activity of recombinant Staphylococcal protein A with introduc... more Aim. 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 immobiliza-tion 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 immu-nosensor. 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. K e y w o r d s: antibodies, recombinant Staphylococcal protein A, protein immobilization, immunosensor, surface plasmon resonance.
Aim. To investigate the formation of an intermediate layer of the immunosensor bioselective eleme... more Aim. 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 im-mobilization of SPA-Cys on a gold surface of the SPR spectrometer while preserving its high immu-noglobulin-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.
Aim. Engineering of recombinant Staphylococcal protein A with cysteine residue (SPA-Cys) for prep... more Aim. 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 af-fi 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 recom-binant 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.
Uploads
Papers by Anna Bakhmachuk