       Document 0591
 DOCN  M9630591
 TI    Expression and purification of the HIV type 1 Rev protein produced in
       Escherichia coli and its use in the generation of monoclonal antibodies.
 DT    9603
 AU    Orsini MJ; Thakur AN; Andrews WW; Hammarskjold ML; Rekosh D; Department
       of Biochemistry, University at Buffalo, New York; 14214, USA.
 SO    AIDS Res Hum Retroviruses. 1995 Aug;11(8):945-53. Unique Identifier :
       AIDSLINE MED/96020095
 AB    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.
 DE    Antibodies, Monoclonal/*IMMUNOLOGY/ISOLATION & PURIF  Base Sequence
       Escherichia coli/GENETICS  Gene Products,
       rev/GENETICS/*IMMUNOLOGY/*ISOLATION & PURIF  Human  Molecular Sequence
       Data  Recombinant Fusion Proteins/GENETICS/IMMUNOLOGY/ISOLATION & PURIF
       JOURNAL ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).

