Fusion protein between protein ZZ and red fluorescent protein DsRed and its application to immunoassays.

Abstract

In the present study, a red fluorescent protein (DsRed) from the coral Discosoma was fused to the C-terminus of protein ZZ, a synthetic artificial IgG-Fc-fragment-binding protein derived from the B-domain of staphylococcal Protein A. The chimaeric protein, tagged with six histidine residues at the N-terminus, was expressed in Escherichia coli and easily purified by one-step Ni2+-chelating affinity chromatography. Its fluorescence and IgG-binding activities were validated using fluorescence-spectrum analysis, ELISA and dot-blot analysis. Furthermore, in subsequent dot-blotting immunoanalysis of glutathione S-transferase and tumour necrosis factor-alpha, and immunofluorescent microscopy assay of interferon regulatory factor 3, the chimaeric protein enabled effective detection of target molecules. Compared with fluorescence-conjugated antibodies, ZZ-DsRed is less susceptible to photobleaching and easy to produce. In addition, unlike HRP (horseradish peroxidase)-conjugated antibodies, using ZZ-DsRed needs no addition of a chromogenic reagent. Our results indicate that ZZ-DsRed shows a wide and promising application potential in immunological detection as a substitute for fluorescent or HRP-conjugated anti-IgGs.

• Authors: Qi-Lai Huang, Cheng Chen, Yun-Zi Chen, Chen-Guang Gong, Jin Wang, Zi-Chun Hua
• Journal: Biotechnology and applied biochemistry (Biotechnol Appl Biochem) Vol. 43 Issue Pt 2 Pg. 121-7 (Feb 2006) ISSN: 0885-4513 [Print] United States
• PMID: 16218907 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Immunoglobulin Fc Fragments
• Immunoglobulin G
• Interferon Regulatory Factor-3
• Luminescent Proteins
• Recombinant Fusion Proteins
• Staphylococcal Protein A
• Tumor Necrosis Factor-alpha
• red fluorescent protein
• Glutathione Transferase

Topics

• Animals
• Chromatography, Affinity
• Cloning, Molecular
• Escherichia coli (metabolism)
• Fluorescent Antibody Technique
• Glutathione Transferase (analysis)
• HeLa Cells
• Humans
• Immunoblotting
• Immunoglobulin Fc Fragments (genetics)
• Immunoglobulin G (genetics)
• Interferon Regulatory Factor-3 (analysis)
• Luminescent Proteins (genetics)
• Microscopy, Fluorescence
• Recombinant Fusion Proteins (genetics, isolation & purification)
• Staphylococcal Protein A (genetics)
• Tumor Necrosis Factor-alpha (analysis)