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An Enzyme-mediated Target-specific Signal Amplifier: Engineered Ascorbate Peroxidase 2/ Antibody-binding Domain Fusion Protein

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Title
An Enzyme-mediated Target-specific Signal Amplifier: Engineered Ascorbate Peroxidase 2/ Antibody-binding Domain Fusion Protein
Author
Lee, Jisu
Advisor
Kang, Sebyung
Keywords
peroxidase; antibody-binding domain; fusion protein; target-specific signal amplifier
Issue Date
2015-02
Publisher
Graduate school of UNIST
Abstract
There are various analytical procedures based on the antigen-antibody interactions and signal amplifications, because antibodies generally give high binding specificity and affinity toward any given target molecules and signal amplification assures the detection of low abundant molecules. We report here the novel approach to substitute conventional horseradish peroxidase (HRP) conjugated secondary antibodies with a recombinant antibody-binding domain (ABD) fused peroxidase. The Z domain, antibody-binding domain of protein A, has high binding affinity and specificity to the Fc region of immunoglobulin G (IgG) and the Engineered ascorbate peroxidase 2 (APEX2) has a capability to catalyze hydrogen peroxides (H2O2) to generate reactive oxygen species which are later utilized for signal amplification. We have genetically fused the antibody binding domain with APEX2 (APEX2-ABD) to give dual functions, antibody binding and peroxidase activity, simultaneously. APEX2-ABD complex is successfully overexpressed and purified with chromatography and characterized with mass spectrometry. Broad binding capability of APEX2-ABD fusion protein to various types of antibodies originated from rabbit, rat, and mouse is confirmed with quartz crystal microbalance (QCM) and surface plasmon resonance (SPR) analyses and the peroxidase activity of APEX2-ABD fusion protein is verified with Amplex red assay. Finally, we apply APEX2-ABD complex as a substitute of conventional HRP-conjugated secondary antibody in the tyramide signal amplification (TSA) detection and we observe significant signal enhancement in cell and tissue imagings.
Description
Department of Biological Sciences
URI
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BIO_Theses_Master
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