Fusion protein between protein ZZ and red fluorescent protein DsRed and its application to immunoassays.
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.
Huang
Qi-Lai
QL
The State Key Laboratory of Pharmaceutical Biotechnology and Department of Biochemistry, College of Life Sciences, Nanjing University, Nanjing 210093, People's Republic of China.
Chen
Cheng
C
Chen
Yun-Zi
YZ
Gong
Chen-Guang
CG
Wang
Jin
J
Hua
Zi-Chun
ZC
D000818:Animals; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D005982:Glutathione Transferase / Q000032:analysis; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007141:Immunoglobulin Fc Fragments / Q000235:genetics*; D007074:Immunoglobulin G / Q000235:genetics*; D050838:Interferon Regulatory Factor-3 / Q000032:analysis; D008164:Luminescent Proteins / Q000235:genetics*; D008856:Microscopy, Fluorescence; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification*; D013205:Staphylococcal Protein A / Q000235:genetics*; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis; D000097573:Red Fluorescent Protein
D005982:Glutathione Transferase; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D050838:Interferon Regulatory Factor-3; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D014409:Tumor Necrosis Factor-alpha; D000097573:Red Fluorescent Protein