Insights into the genetic re-engineering of chimeric antibody-binding green fluorescent proteins for immunological taggers

Genetic re-manipulation of chimeric antibody-binding green fluorescent proteins was successfully conducted to create versatile tools for immunological diagnosis. Four chimeric GFPs carrying one and two-consecutive sequences of the Fc-binding motif (Z-domain), derivative of IgG-binding B domain of Staphylococcal protein A (SpA), at the C-terminus were constructed. The chimeric Ab-binding GFPs possessed dual characteristics of both IgG-binding and activity of fluorescent emission. The chimeric proteins were purified to homogeneity using an IgG-Sepharose column. Additionally, a hexahistidine was fused to the N-terminal of the GFPZ and GFPZZ to allow a high protein recovery obtained from immobilized metal (Ni^2+) affinity chromatography (Ni-NTA), and for protein immobilization to the sensor surface. Results obtained from the Surface Plasmon Resonance (SPR) revealed a high binding affinity (K_a) to immobilized human immunoglobulin up to 6.7 and 81.1 (107/M) for the GFPZ and GFPZZ, respectively. This affinity constant was raised up to 2-5 times higher when the chimeric GFPs harboring hexahistidine residues were captured on the sensor chip via metal coordination. The strong binding affinity to IgG of the chimeric GFPs was clinically applied to detect the antinuclear antibody. A strong intensity of fluorescence, higher than that of the classical fluorescein isothiocyanate (FITC) conjugated system, was significantly detected. Moreover, the proteins with double repeats of Fc-binding motif (GFPZZ and H6GFPZZ) obviously demonstrated a more intense fluorescent signal than those of the single Z domain, which corresponded to the result from SPR. All these findings support a high potential for applying such chimeric Ab-binding GFPs for clinical applications

Chimeric Antibody-Binding Vitreoscilla Hemoglobin (VHb) Mediates Redox-Catalysis Reaction: New Insight into the Functional Role of VHb

Experimentation was initiated to explore insight into the redox-catalysis reaction derived from the heme prosthetic group of chimeric Vitreoscilla hemoglobin (VHb). Two chimeric genes encoding chimeric VHbs harboring one and two consecutive sequences of Fc-binding motif (Z-domain) were successfully constructed and expressed in E. coli strain TG1. The chimeric ZVHb and ZZVHb were purified to a high purity of more than 95% using IgG-Sepharose affinity chromatography. From surface plasmon resonance, binding affinity constants of the chimeric ZVHb and ZZVHb to human IgG were 9.7 x 10(7) and 49.1 x 10(7) per molar, respectively. More importantly, the chimeric VHbs exhibited a peroxidase-like activity determined by activity staining on native PAGE and dot blotting. Effects of pH, salt, buffer system, level of peroxidase substrate and chromogen substrate were determined in order to maximize the catalytic reaction. From our findings, the chimeric VHbs displayed their maximum peroxidase-like activity at the neutral pH (~7.0) in the presence of high concentration (20-40 mM) of hydrogen peroxide. Under such conditions, the detection limit derived from the calibration curve was at 250 ng for the chimeric VHbs, which was approximately 5-fold higher than that of the horseradish peroxidase. These findings reveal the novel functional role of Vitreoscilla hemoglobin indicating a high trend of feasibility for further biotechnological and medical applications

Exploring Matrix Effects on Binding Properties and Characterization of Cotinine Molecularly Imprinted Polymer on Paper-Based Scaffold

Commercially available sorbent materials for solid-phase extraction are widely used in analytical laboratories. However, non-selective binding is a major obstacle for sample analysis. To overcome this problem, molecularly imprinted polymers (MIPs) were used as selective adsorbent materials prior to determining target analysts. In this study, the use of non-covalent molecularly imprinted polymers (MIPs) for cotinine adsorption on a paper-based scaffold was studied. Fiberglass paper was used as a paper scaffold for cotinine-selective MIP adsorption with the use of 0.5% agarose gel. The effects of salt, pH, sample matrix, and solvent on the cotinine adsorption and extraction process were investigated. Under optimal conditions, the adsorption isotherm of synthesized MIPs increased to 125.41 µg/g, whereas the maximum adsorption isotherm of non-imprinted polymers (NIPs) was stable at 42.86 µg/g. The ability of the MIP paper scaffold to absorb cotinine in water medium was approximately 1.8–2.8-fold higher than that of the NIP scaffold. From Scatchard analysis, two dissociation constants of MIPs were calculated to be 2.56 and 27.03 µM. Nicotine, myosmine, and N-nitrosonornicotine were used for selectivity testing, and the calculated selectivity factor of cotinine to nicotine, myosmine, and N-nitrosonornicotine was 1.56, 2.69, and 2.05, respectively. Overall, the MIP paper scaffold is promising for simple onsite sampling of cotinine and can be used to assess tobacco smoke exposure

Chimeric Antibody-Binding Vitreoscilla Hemoglobin (VHb) Mediates Redox-Catalysis Reaction: New Insight into the Functional Role of VHb

<p>Experimentation was initiated to explore insight into the redox-catalysis reaction derived from the heme prosthetic group of chimeric <i>Vitreoscilla</i> hemoglobin (VHb). Two chimeric genes encoding chimeric VHbs harboring one and two consecutive sequences of Fc-binding motif (Z-domain) were successfully constructed and expressed in <i>E. coli</i> strain TG1. The chimeric ZVHb and ZZVHb were purified to a high purity of more than 95% using IgG-Sepharose affinity chromatography. From surface plasmon resonance, binding affinity constants of the chimeric ZVHb and ZZVHb to human IgG were 9.7 x 10⁷ and 49.1 x 10⁷ per molar, respectively. More importantly, the chimeric VHbs exhibited a peroxidase-like activity determined by activity staining on native PAGE and dot blotting. Effects of pH, salt, buffer system, level of peroxidase substrate and chromogen substrate were determined in order to maximize the catalytic reaction. From our findings, the chimeric VHbs displayed their maximum peroxidase-like activity at the neutral pH (&#126;7.0) in the presence of high concentration (20-40 mM) of hydrogen peroxide. Under such conditions, the detection limit derived from the calibration curve was at 250 ng for the chimeric VHbs, which was approximately 5-fold higher than that of the horseradish peroxidase. These findings reveal the novel functional role of <i>Vitreoscilla</i> hemoglobin indicating a high trend of feasibility for further biotechnological and medical applications.</p

Co-expression of zinc binding motif and GFP as a cellular indicator of metal ions mobility-3

<p><b>Copyright information:</b></p><p>Taken from "Co-expression of zinc binding motif and GFP as a cellular indicator of metal ions mobility"</p><p>International Journal of Biological Sciences 2005;1(4):146-151.</p><p>Published online 1 Dec 2005</p><p>PMCID:PMC1345397.</p><p>© Ivyspring International Publisher. This is an open access article. Reproduction is permitted for personal and noncommerical use, provided that the article is in whole, unmodified, and properly cited.</p>cellularly (TG1/pHis6GFPuv) (a) and engineered cells co-expressing zinc-binding motif on the cell surface and His6GFP in the cytoplasm (TG1/pEVZn + pHis6GFPuv) (b