Recombinant Staphylococcal protein A with cysteine residue for preparation of affinity chromatography stationary phase and immunosensor applications

Abstract

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.

Surface Plasmon Resonance Investigations of Bioselective Element Based on the Recombinant Protein A for Immunoglobulin Detection

“…Synthesis and purification of recombinant Staphylococcal protein A with specially introduced C-terminal cysteine residue (SPA-Cys) were described in [ 25 ]. The homogeneity of the proteins used was checked by electrophoresis in 13% polyacrylamide gel under denaturing conditions.…”

“…Moreover, this variant of immobilization of SPA improves the IgG-binding activity of SPA as well as further antigen-binding activity of antibody molecules immobilized through SPA [ 24 ]. In our previous work, the original plasmid pET24-SPA-6HisCys was constructed, the recombinant protein A contained all five IgG-binding domains, 6His-tag and C-terminal cysteine residue (SPA-Cys) was obtained, and its immobilization on the gold sensor surface was demonstrated [ 25 ].…”

Study on interactions of human IgG with immobilized anti-IgG or recombinant Staphylococcal protein A using surface plasmon resonance spectrometry

“…Human IgG was obtained also by affinity chromatography using SPA-Cys-modified silica as described in [9]. The synthesis and purification of recombinant SPA-Cys were described in [9]. The homogeneity of the proteins used was checked by 13% SDS-PAGE.…”

“…Genetically engineered introduction into the recombinant SPA of the cysteine residue, which interacts with the gold sensor surface through exposed SH-group, improves the reliability of SPA immobilization preserving its functional properties. In the Institute of Molecular Biology and Genetics NAS of Ukraine, the original genetically engineered construct was created and the recombinant Staphylococcal protein A with the introduced C-terminal cysteine residue (SPA-Cys) was obtained [9]. Species-specific antibodies, such as goat anti-human IgG antibodies (anti-IgG), are more traditional immunoglobulin-binding proteins, which could also be immobilized on a sensor surface as an intermediate layer for further immobilization of IgG.…”

Study on efficiency of oriented immobilization of antibodies on the SPR sensor surface using Staphylococcal protein A or its recombinant analogue

“…Genetically engineered introduction of one cysteine residue into recombinant SPA, which interacts with the gold sensor surface through exposed SH-group, improves reliability of the SPA immobilization and accessibility of IgG-binding sites [8]. In the Institute of Molecular Biology and Genetics of the NAS of Ukraine, an original genetically engineered construct was created and a recombinant Staphylococcal protein A with specially introduced C-terminal cysteine residue (SPA-Cys) was obtained [9].…”