Application to immunoassays of the fusion protein between protein ZZ and enhanced green fluorescent protein

• Published in: Journal of immunological methods Vol. 309; no. 1; pp. 130 - 138
• Main Authors: Huang, Qi-Lai, Chen, Cheng, Chen, Yun-Zi, Gong, Chen-Guang, Cao, Lin, Wang, Jin, Hua, Zi-Chun
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 20.02.2006; Elsevier
• Subjects: Aequorea victoria; Amino Acid Sequence; Base Sequence; Biological and medical sciences; Blotting, Western; Cell Line; Cell Separation; DNA, Recombinant - genetics; Dot blotting; EGFP; Enzyme-Linked Immunosorbent Assay; Escherichia coli; Flow Cytometry; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Fusion; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - isolation & purification; Humans; Immunoassay - methods; Immunoblotting; Immunofluorescent microscopy assay; Immunoglobulin Fc Fragments - genetics; Immunoglobulin Fc Fragments - isolation & purification; Immunoglobulin G - metabolism; Ligands; Microscopy, Fluorescence; Molecular immunology; Molecular Sequence Data; Protein Binding; Protein ZZ; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - isolation & purification; Techniques; Tumor Necrosis Factor-alpha - analysis; Protein ZZ; Dot blotting; EGFP; Fusion; Immunofluorescent microscopy assay; Microscopy; Green fluorescent protein; Fusion protein; Immunological method
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/j.jim.2005.11.009

Enhanced green fluorescent protein (EGFP) from Aequorea victoria was fused to the C terminal region of protein ZZ, an artificial synthetic IgG Fc fragment binding protein derived from tandem repeats of the B domain of protein A. The ZZ–EGFP fusion protein was expressed in Escherichia coli with a His 6 tag and purified in high yield by one-step Ni 2+ chelating affinity chromatography. It was then used in the immunoblot analysis of GST and TNFα as well as in immunofluorescent assays of 293T cells transfected with IRF3, an interferon regulatory factor which localized in cytoplasm without virus infection. The fusion protein also performed effectively in FACS analysis of surface integrin β3 subunit on 293 T cells. The chimeric protein bound various antibodies from different animal sources, directed against a variety of proteins. Thus, ZZ–EGFP showed broad promise in potential immunological applications.