DISSECTION OF THE EXTRACELLULAR HUMAN INTERFERON-GAMMA RECEPTOR ALPHA-CHAIN INTO 2 IMMUNOGLOBULIN-LIKE DOMAINS - PRODUCTION IN AN ESCHERICHIA-COLI THIOREDOXIN GENE FUSION EXPRESSION SYSTEM AND RECOGNITION BY NEUTRALIZING ANTIBODIES

The extracellular interferon gamma receptor alpha-chain (IFN gamma R) is believed to comprise two discrete approximate to 110 amino acid imm unoglobulin-like domains, perhaps similar to those seen in the crystal structure of the extracellular human growth hormone receptor [De Vos, A. M., Ultsch, M., and Kossiakoff, A. (1992) Science 255, 306-312], a distant relative in the cytokine receptor superfamily. In accord with this idea, we show that these IFN gamma R immunoglobulin-like domains can be produced separately in a soluble form with a native-like fold. The N-terminal domain (residues 1-108), with a Cys(105) to Ser(105) m utation, was produced at a high level, in a soluble form, as a thiored oxin-interferon gamma receptor fragment fusion protein in the cytoplas m of Escherichia coli. Upon extraction, the receptor Cys(60)-Cys(68) d isulfide bond formed spontaneously, to generate a native-like structur e directly without the need for refolding. Cleavage of the fusion prot ein by enterokinase released the receptor fragment (approximate to 12 kDa), which was recognized by several neutralizing antibodies with aff inities, measured using surface plasmon resonance technology, that wer e essentially indistinguishable from those seen with the full length e xtracellular IFN gamma R produced in eukaryotic cells. Circular dichro ism and 1D H-1 nuclear magnetic resonance spectra indicated that the r eceptor fragment adopts a folded state, with mainly beta-sheet and rev erse turn secondary structure. The second membrane-proximal Ig-like do main of the IFN gamma R (residues 90-229) was produced, albeit less ef ficiently, and characterized in a similar way. The production of these two independently folded proteins provides experimental support for t he two domain organization of the IFN gamma R and opens new avenues fo r structural studies on these Ig-like molecules by NMR and crystallogr aphic methods.