SOLUBLE FASR LIGAND-BINDING DOMAIN - HIGH-YIELD PRODUCTION OF ACTIVE FUSION AND NON-FUSION RECOMBINANT PROTEINS USING THE BACULOVIRUS INSECT CELL SYSTEM/

We used the recombinant baculovirus/insect cell system to express two soluble forms of the mouse Fas receptor (mFasR) extracellular domain ( ECD): a monomer comprising the entire ligand-binding portion of mFasR followed by a carboxy-terminal hexa-histidine extension aiding purific ation by immobilized metal affinity chromatography and an immunoadhesi n in which the same 148 residues were fused to the Fc portion of a tru ncated human IgG1 immunoglobulin heavy chain, Both constructs harboure d a 24 base pairs insertion placed upstream of the initiating ATG [Pea kman, Charles, Sydenham, Gewert, Page, and Makoff (1992) Nucleic Acids Res. 20, 6111-6112]. Despite its hexa-histidine extension, the monova lent recombinant protein from crude culture media failed to bind immob ilized Ni2+ unless proteins were first precipitated twice by ammonium sulphate, The overall procedure then yielded approximate to 10 mg/l of protein which could be purified to near homogeneity using two additio nal chromatographic steps. The glycosylated polypeptide migrated as a band of M-r = (21-31) x 10(3) in SDS/PAGE and was monomeric in physiol ogical buffers. Under non-reducing conditions, denaturation in 6 M gua nidinium chloride was reversible after slow removal of the denaturing agent, The mFasR immuno-adhesin was secreted (approximate to 5-10 mg/l ) as a disulphide-linked homodimer, and endowed with ligand-binding ac tivity since it could bind Fast on the surface of D11S, Fast-expressin g cells. When tested for their ability to inhibit FasR-dependent cell lysis, the soluble dimeric immunoadhesin markedly inhibited FasL-media ted cytotoxicity (IC50 approximate to 30 nM), and was approximate to 6 times as effective as its monomeric counterpart.