Dissection of the extracellular human interferon gamma receptor alpha-chain into two immunoglobulin-like domains. Production in an Escherichia coli thioredoxin gene fusion expression system and recognition by neutralizing antibodies.

Details

Serval ID
serval:BIB_8F9D9BC4082E
Type
Article: article from journal or magazin.
Collection
Publications
Title
Dissection of the extracellular human interferon gamma receptor alpha-chain into two immunoglobulin-like domains. Production in an Escherichia coli thioredoxin gene fusion expression system and recognition by neutralizing antibodies.
Journal
Biochemistry
Author(s)
Williams G., Ruegg N., Birch A., Weber C., Hofstädter K., Robinson J.A., Aguet M., Garotta G., Schlatter D., Huber W.
ISSN
0006-2960 (Print)
ISSN-L
0006-2960
Publication state
Published
Issued date
1995
Volume
34
Number
5
Pages
1787-1797
Language
english
Abstract
The extracellular interferon gamma receptor alpha-chain (IFN gamma R) is believed to comprise two discrete approximately 110 amino acid immunoglobulin-like domains, perhaps similar to those seen in the crystal structure of the extracellular human growth hormone receptor [De Vos, A. M., Ultsch, M., & 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 Cys105 to Ser105 mutation, was produced at a high level, in a soluble form, as a thioredoxin-interferon gamma receptor fragment fusion protein in the cytoplasm of Escherichia coli. Upon extraction, the receptor Cys60-Cys68 disulfide bond formed spontaneously, to generate a native-like structure directly without the need for refolding. Cleavage of the fusion protein by enterokinase released the receptor fragment (approximately 12 kDa), which was recognized by several neutralizing antibodies with affinities, measured using surface plasmon resonance technology, that were essentially indistinguishable from those seen with the full length extracellular IFN gamma R produced in eukaryotic cells. Circular dichroism and 1D 1H nuclear magnetic resonance spectra indicated that the receptor fragment adopts a folded state, with mainly beta-sheet and reverse turn secondary structure. The second membrane-proximal Ig-like domain of the IFN gamma R (residues 90-229) was produced, albeit less efficiently, and characterized in a similar way. The production of these two independently folded proteins provides experimental support for the two domain organization of the IFN gamma R and opens new avenues for structural studies on these Ig-like molecules by NMR and crystallographic methods.
Keywords
Amino Acid Sequence, Antibodies, Base Sequence, Cloning, Molecular, Escherichia coli/genetics, Humans, Magnetic Resonance Spectroscopy, Molecular Sequence Data, Mutagenesis, Site-Directed, Plasmids, Polymerase Chain Reaction, Receptors, Interferon/chemistry, Receptors, Interferon/genetics, Thioredoxins/genetics
Pubmed
Web of science
Create date
28/01/2008 12:36
Last modification date
20/08/2019 15:53
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