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Titolo:
EXPRESSION AND PURIFICATION OF THE HIV TYPE-1 REV PROTEIN PRODUCED INESCHERICHIA-COLI AND ITS USE IN THE GENERATION OF MONOCLONAL-ANTIBODIES
Autore:
ORSINI MJ; THAKUR AN; ANDREWS WW; HAMMARSKJOLD ML; REKOSH D;
Indirizzi:
UNIV VIRGINIA,MYLES H THALER CTR AIDS & HUMAN RETROVIRUS RES,HSC BOX 441 CHARLOTTESVILLE VA 22908 SUNY BUFFALO,DEPT BIOCHEM BUFFALO NY 14214 SUNY BUFFALO,DEPT MICROBIOL BUFFALO NY 14214
Titolo Testata:
AIDS research and human retroviruses
fascicolo: 8, volume: 11, anno: 1995,
pagine: 945 - 953
SICI:
0889-2229(1995)11:8<945:EAPOTH>2.0.ZU;2-5
Fonte:
ISI
Lingua:
ENG
Soggetto:
HUMAN-IMMUNODEFICIENCY-VIRUS; LATE REPLACEMENT VECTOR; VIRAL MESSENGER-RNA; TRANS-ACTIVATOR; TARGET SEQUENCE; GENE-EXPRESSION; I REX; NUCLEOLAR LOCALIZATION; MUTATIONAL ANALYSIS; RESPONSIVE ELEMENT;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Science Citation Index Expanded
Citazioni:
57
Recensione:
Indirizzi per estratti:
Citazione:
M.J. Orsini et al., "EXPRESSION AND PURIFICATION OF THE HIV TYPE-1 REV PROTEIN PRODUCED INESCHERICHIA-COLI AND ITS USE IN THE GENERATION OF MONOCLONAL-ANTIBODIES", AIDS research and human retroviruses, 11(8), 1995, pp. 945-953

Abstract

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 betweenamino 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 furtherstudies of Rev function.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 25/11/20 alle ore 06:38:27