Authors: Bülow, Leif
Isarankura-Na-Ayudhya, Chartchalerm
Prachayasittikul, Virapong
Suwanwong, Yaneenart
Title: Insights into the genetic re-engineering of chimeric antibody-binding green fluorescent proteins for immunological taggers
Language (ISO): en
Abstract: 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.
Subject Headings: Chimeric Antibody-binding GFP
C-terminus GFP fusion
Fluorescent antinuclear antibody analysis
Surface Plasmon Resonance
Issue Date: 2006-10-16
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