Expression and biological activity of two recombinant polypeptides related to subunit 1 of the interferon-alpha receptor
Braz. j. med. biol. res
; 33(7): 771-8, July 2000. ilus, graf
Article
em En
| LILACS
| ID: lil-262676
Biblioteca responsável:
BR1.1
RESUMO
Abnormal production of interferon alpha (IFN-a) has been found in certain autoimmune diseases and can be also observed after prolonged therapy with IFN-a. IFN-a can contribute to the pathogenesis of allograft rejection in bone marrow transplants. Therefore, the development of IFN-a inhibitors as a soluble receptor protein may be valuable for the therapeutic control of these diseases. We have expressed two polypeptides encoding amino acids 93-260 (P1) and 261-410 (P2) of the extracellular domain of subunit 1 of the interferon-a receptor (IFNAR 1-EC) in E. coli. The activities of the recombinant polypeptides and of their respective antibodies were evaluated using antiproliferative and antiviral assays. Expression of P1 and P2 polypeptides was achieved by transformation of cloned plasmid pRSET A into E. coli BL21(DE3)pLysS and by IPTG induction. P1 and P2 were purified by serial sonication steps and by gel filtration chromatography with 8 M urea and refolded by dialysis. Under reducing SDS-PAGE conditions, the molecular weight of P1 and P2 was 22 and 17 kDa, respectively. Polyclonal anti-P1 and anti-P2 antibodies were produced in mice. P1 and P2 and their respective polyclonal antibodies were able to block the antiproliferative activity of 6.25 nM IFN-aB on Daudi cells, but did not block IFN-aB activity at higher concentrations (>6.25 nM). On the other hand, the polypeptides and their respective antibodies did not inhibit the antiviral activity of IFN-aB on Hep 2/c cells challenged with encephalomyocarditis virus.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
LILACS
Assunto principal:
Antivirais
/
Peptídeos
/
Interferon Tipo I
/
Interferon-alfa
/
Receptores de Interferon
/
Escherichia coli
Limite:
Animals
/
Humans
Idioma:
En
Revista:
Braz. j. med. biol. res
Assunto da revista:
BIOLOGIA
/
MEDICINA
Ano de publicação:
2000
Tipo de documento:
Article
País de afiliação:
Brasil
País de publicação:
Brasil