Expression and purification of the HIV type 1 Rev protein produced in Escherichia coli and its use in the generation of monoclonal antibodies.
We have developed a simple and rapid procedure for the purification of large amounts of Rev protein overexpressed in E. coli. The purification method, which does not require denaturation of the protein, takes advantage of the positively charged nature of Rev and the ability of Rev to interact with nucleic acids. The purified protein was used to develop three novel murine monoclonal antibodies against Rev. Using fusion proteins between glutathione S-transferase (GST) and various fragments of the Rev protein, we mapped the specificity of these antibodies to different regions of the Rev protein. One antibody, 3H6, is directed against the nucleolar localization/RRE-binding domain of Rev between amino acids 38 and 44. Another antibody, 3G4, recognizes an epitope between amino acids 90 and 116 of Rev. A third antibody, 2G2, does not recognize any of the fusion proteins, and may be directed against a conformational epitope. All three antibodies are able to detect Rev on Western blots and to immunoprecipitate Rev under native conditions. However, only 3H6 and 3G4 immunoprecipitate Rev under denaturing conditions and are able to detect Rev expressed in transfected cells by indirect immunofluorescence. These antibodies should prove useful in further studies of Rev function.
D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000302:isolation & purification; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D015694:Gene Products, rev / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification*; D006801:Humans; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D015694:Gene Products, rev; D011993:Recombinant Fusion Proteins