Expression and biological activity of two recombinant polypeptides relatedto subunit 1 of the interferon-alpha receptor

Abnormal production of interferon alpha (IFN-alpha) has been found in certa in autoimmune diseases and can be also observed after prolonged therapy wit h IFN-alpha, IFN-alpha can contribute to the pathogenesis of allograft reje ction in bone marrow transplants. Therefore, the development of IFN-alpha i nhibitors as a soluble receptor protein may be valuable for the therapeutic control of these diseases. We have expressed two polypeptides encoding ami no acids 93-260 (P1) and 261-410 (P2) of the extracellular domain of subuni t 1 of the interferon-alpha receptor (IFNAR 1-EC) in E. coli. The activitie s of the recombinant polypeptides and of their respective antibodies were e valuated using antiproliferative and antiviral assays. Expression of P1 and P2 polypeptides was achieved by transformation of cloned plasmid pRSET A i nto E. coli BL21(DE3)pLysS and by IPTG induction. P1 and P2 were purified b y serial sonication steps and by gel filtration chromatography with 8 M ure a and refolded by dialysis. Under reducing SDS-PAGE conditions, the molecul ar weight of P1 and P2 was 22 and 17 kDa, respectively, Polyclonal anti-P1 and anti-P2 antibodies were produced in mice. Pi and P2 and their respectiv e polyclonal antibodies were able to block the antiproliferative activity o f 6.25 nM IFN-alpha beta on Daudi cells, but did not block IFN-alpha beta a ctivity at higher concentrations (>6.25 nM). On the other hand, the polypep tides and their respective antibodies did not inhibit the antiviral activit y of IFN-alpha beta on Hep 2/c cells challenged with encephalomyocarditis v irus.