Expression and biological activity of two recombinant polypeptides related to subunit 1 of the interferon-a receptor
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 s...
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| Format: | Article |
| Language: | English |
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Associação Brasileira de Divulgação Científica
2000-07-01
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| Series: | Brazilian Journal of Medical and Biological Research |
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| Online Access: | http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2000000700007 |
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| author | S. Yoon R.D.C. Hirata N.Y. Nguyen R. Curi M. Russo M.H. Hirata |
| author_facet | S. Yoon R.D.C. Hirata N.Y. Nguyen R. Curi M. Russo M.H. Hirata |
| author_sort | S. Yoon |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-12T16:48:50Z |
| format | Article |
| id | doaj.art-5a4677a8604b4dd6947cc27fa71ad0c3 |
| institution | Directory Open Access Journal |
| issn | 0100-879X 1414-431X |
| language | English |
| last_indexed | 2024-12-12T16:48:50Z |
| publishDate | 2000-07-01 |
| publisher | Associação Brasileira de Divulgação Científica |
| record_format | Article |
| series | Brazilian Journal of Medical and Biological Research |
| spelling | doaj.art-5a4677a8604b4dd6947cc27fa71ad0c32022-12-22T00:18:25ZengAssociação Brasileira de Divulgação CientíficaBrazilian Journal of Medical and Biological Research0100-879X1414-431X2000-07-0133777177810.1590/S0100-879X2000000700007Expression and biological activity of two recombinant polypeptides related to subunit 1 of the interferon-a receptorS. YoonR.D.C. HirataN.Y. NguyenR. CuriM. RussoM.H. HirataAbnormal 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.http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2000000700007interferon alphainterferon-a receptorinhibitor of interferon-aautoimmune diseasesgraft-versus-host diseaserecombinant protein |
| spellingShingle | S. Yoon R.D.C. Hirata N.Y. Nguyen R. Curi M. Russo M.H. Hirata Expression and biological activity of two recombinant polypeptides related to subunit 1 of the interferon-a receptor Brazilian Journal of Medical and Biological Research interferon alpha interferon-a receptor inhibitor of interferon-a autoimmune diseases graft-versus-host disease recombinant protein |
| title | Expression and biological activity of two recombinant polypeptides related to subunit 1 of the interferon-a receptor |
| title_full | Expression and biological activity of two recombinant polypeptides related to subunit 1 of the interferon-a receptor |
| title_fullStr | Expression and biological activity of two recombinant polypeptides related to subunit 1 of the interferon-a receptor |
| title_full_unstemmed | Expression and biological activity of two recombinant polypeptides related to subunit 1 of the interferon-a receptor |
| title_short | Expression and biological activity of two recombinant polypeptides related to subunit 1 of the interferon-a receptor |
| title_sort | expression and biological activity of two recombinant polypeptides related to subunit 1 of the interferon a receptor |
| topic | interferon alpha interferon-a receptor inhibitor of interferon-a autoimmune diseases graft-versus-host disease recombinant protein |
| url | http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2000000700007 |
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