Application to immunoassays of the fusion protein between protein ZZ and enhanced green fluorescent protein.
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 TNFalpha 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 beta3 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.
Huang
Qi-Lai
QL
The State Key Laboratory of Pharmaceutical Biotechnology and Department of Biochemistry, College of Life Sciences, Nanjing University, 22 Hankou Road, Nanjing 210093, P.R. China.
Chen
Cheng
C
Chen
Yun-Zi
YZ
Gong
Chen-Guang
CG
Cao
Lin
L
Wang
Jin
J
Hua
Zi-Chun
ZC
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D002469:Cell Separation; D004274:DNA, Recombinant / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D015151:Immunoblotting; D007141:Immunoglobulin Fc Fragments / Q000235:genetics* / Q000302:isolation & purification; D007074:Immunoglobulin G / Q000378:metabolism; D008024:Ligands; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis
D004274:DNA, Recombinant; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D008024:Ligands; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins