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 purification by immobilized metal affinity chromatography and an immunoadhesin in which the same 148 residues were fused to the Fc portion of a truncated human IgG1 immunoglobulin heavy chain. Both constructs harboured a 24 base pairs insertion placed upstream of the initiating ATG [Peakman, Charles, Sydenham, Gewert, Page, and Makoff (1992) Nucleic Acids Res. 20, 6111-6112]. Despite its hexa-histidine extension, the monovalent recombinant protein from crude culture media failed to bind immobilized Ni2+ unless proteins were first precipitated twice by ammonium sulphate. The overall procedure then yielded ≈ 10 mg/l of protein which could be purified to near homogeneity using two additional chromatographic steps. The glycosylated polypeptide migrated as a band of Mr = (21-31)×103 in SDS/PAGE and was monomeric in physiological buffers. Under non-reducing conditions, denaturation in 6 M guanidinium chloride was reversible after slow removal of the denaturing agent. The mFasR immunoadhesin was secreted (≈ 5-10 mg/l) as a disulphide-linked homodimer, and endowed with ligand-binding activity since it could bind FasL on the surface of D11S, FasL-expressing cells. When tested for their ability to inhibit FasR-dependent cell lysis, the soluble dimeric immunoadhesin markedly inhibited FasL-mediated cytotoxicity (IC50 ≈ 30 nM), and was ≈ 6 times as effective as its monomeric counterpart.
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Research Article|
March 01 1998
Soluble FasR ligand-binding domain: high-yield production of active fusion and non-fusion recombinant proteins using the baculovirus/insect cell system
Jérôme MAHIOU;
Jérôme MAHIOU
*INSERM U 373, Faculté de Médecine Necker, 156 rue de Vaugirard, 75730 Paris Cedex 15, France
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Jean-Pierre ABASTADO;
Jean-Pierre ABASTADO
†INSERM U 277, Biologie Moléculaire du Gène, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France
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Lucien CABANIE;
Lucien CABANIE
‡UMR 144, Institut Curie, 12 rue Lhomond, 75005 Paris, France
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François GODEAU
François GODEAU
1
*INSERM U 373, Faculté de Médecine Necker, 156 rue de Vaugirard, 75730 Paris Cedex 15, France
1To whom correspondence should be addressed. Present address: INSERM U 267, Hôpital Paul Brousse, 12-14, avenue Paul Vaillant-Couturier, 94807 Villejuif Cedex, France.
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Publisher: Portland Press Ltd
Received:
September 29 1997
Revision Received:
November 11 1997
Accepted:
November 28 1997
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1998
1998
Biochem J (1998) 330 (2): 1051–1058.
Article history
Received:
September 29 1997
Revision Received:
November 11 1997
Accepted:
November 28 1997
Citation
Jérôme MAHIOU, Jean-Pierre ABASTADO, Lucien CABANIE, François GODEAU; Soluble FasR ligand-binding domain: high-yield production of active fusion and non-fusion recombinant proteins using the baculovirus/insect cell system. Biochem J 1 March 1998; 330 (2): 1051–1058. doi: https://doi.org/10.1042/bj3301051
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