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Fusion protein between protein ZZ and red fluorescent protein DsRed and its application to immunoassays
Author(s) -
Huang QiLai,
Chen Cheng,
Chen YunZi,
Gong ChenGuang,
Wang Jin,
Hua ZiChun
Publication year - 2006
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1042/ba20050136
Subject(s) - fusion protein , horseradish peroxidase , microbiology and biotechnology , affinity chromatography , protein a , chemistry , protein g , biochemistry , protein a/g , protein tag , myc tag , fluorescence , green fluorescent protein , chromogenic , biology , antibody , recombinant dna , chromatography , enzyme , physics , immunology , gene , quantum mechanics
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 Ni 2+ ‐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‐α, 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.