Expression and biological activity of two recombinant polypeptides related to subunit 1 of the interferon-alpha receptor.
Abnormal production of interferon alpha (IFN-alpha) has been found in certain autoimmune diseases and can be also observed after prolonged therapy with IFN-alpha. IFN-alpha can contribute to the pathogenesis of allograft rejection in bone marrow transplants. Therefore, the development of IFN-alpha 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-alpha 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-alphaB on Daudi cells, but did not block IFN-alphaB 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-alphaB on Hep 2/c cells challenged with encephalomyocarditis virus.
Yoon
S
S
Departamento de Análises Clínicas e Toxicológicas, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, SP, Brasil. sunok@zipmail.com.br
Hirata
R D
RD
Nguyen
N Y
NY
Curi
R
R
Russo
M
M
Hirata
M H
MH
D000818:Animals; D000998:Antiviral Agents / Q000037:antagonists & inhibitors*; D002417:Cattle; D004926:Escherichia coli / Q000737:chemistry*; D006801:Humans; D007370:Interferon Type I / Q000037:antagonists & inhibitors*; D016898:Interferon-alpha / Q000037:antagonists & inhibitors* / Q000378:metabolism; D051379:Mice; D010446:Peptide Fragments / Q000378:metabolism / Q000494:pharmacology*; D017471:Receptors, Interferon / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins; D018709:Statistics, Nonparametric; D014764:Viral Proteins / Q000737:chemistry*
D000998:Antiviral Agents; D007370:Interferon Type I; D016898:Interferon-alpha; D010446:Peptide Fragments; D017471:Receptors, Interferon; D011994:Recombinant Proteins; D014764:Viral Proteins