A novel EBNA-1 tag system for high level expression and efficient detection of fusion proteins in vitro and in vivo.
A highly specific monoclonal antibody, which recognises an epitope between amino acids (aa) 408 and 446 of EBNA-1, was found to immunoprecipitate this protein with great efficiency. By adding a 39-aa tag, the BGLF4 protein product of EBV was shown to express in the cytoplasm of transfected cells. This EBNA-1 tag could be used for the detection of specific protein expression by immunoblotting, immunofluorescence and, especially, immunoprecipitation assays. A plasmid vector encoding this EBNA-1 tag sequence was therefore designed for efficient expression both in vitro and in vivo. The efficient translational start signal of black beetle virus (BBV) was placed under the control of the SV40 or T7 promoters, and the beta-globin splicing signal used to enhance the transportation of mRNA from the nucleus to the cytoplasm.
Chen
M R
MR
Graduate Institute of Microbiology, College of Medicine, National Taiwan University, Taipei, ROC. mrc@ha.mc.ntu.edu.tw
Huang
H
H
Fen
C Y
CY
Chen
J Y
JY
D000911:Antibodies, Monoclonal; D002460:Cell Line; D000939:Epitopes; D019309:Epstein-Barr Virus Nuclear Antigens / Q000276:immunology / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors; D006801:Humans; D015151:Immunoblotting; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D014764:Viral Proteins
D000911:Antibodies, Monoclonal; D000939:Epitopes; D019309:Epstein-Barr Virus Nuclear Antigens; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C406063:BGLF4 protein, Epstein-Barr virus; D017346:Protein Serine-Threonine Kinases