Purification and biochemical characterization of a soluble human interferon gamma receptor expressed in Escherichia coli.

Fountoulakis, Michael; Juranville, Jean‐François; Stüber, Dietrich; Weibel, E. K.; Garotta, Gianni

doi:10.1016/s0021-9258(19)38294-8

Cited by 55 publications

(6 citation statements)

References 20 publications

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“…To further characterize intramolecular disulfide bond formation and metal binding resulting from copper addition, we turned to native SDS–PAGE. Proteins that remain more compact through conformational trapping − have been observed to migrate faster through a gel than their native counterparts. Metal binding can also exert a similar effect, whereby the extent of protein unfolding is limited by the geometry of the bound metal .…”

Section: Resultsmentioning

confidence: 99%

Human γS-Crystallin–Copper Binding Helps Buffer against Aggregation Caused by Oxidative Damage

et al. 2020

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Divalent metal cations can play a role in protein aggregation diseases, including cataract. Here we compare the aggregation of human γS-crystallin, a key structural protein of the eye lens, via mutagenesis, ultraviolet light damage, and the addition of metal ions. All three aggregation pathways result in globular, amorphous-looking structures that do not elongate into fibers. We also investigate the molecular mechanism underlying copper(II)induced aggregation. This work was motivated by the observation that zinc(II)-induced aggregation of γS-crystallin is driven by intermolecular bridging of solvent-accessible cysteine residues, while in contrast, copper(II)-induced aggregation of this protein is exacerbated by the removal of solvent-accessible cysteines via mutation. Here we find that copper(II)-induced aggregation results from a complex mechanism involving multiple interactions with the protein. The initial protein−metal interactions result in the reduction of Cu(II) to Cu(I) with concomitant oxidation of γScrystallin. In addition to the intermolecular disulfides that represent a starting point for aggregation, intramolecular disulfides also occur in the cysteine loop, a region of the N-terminal domain that was previously found to mediate the early stages of cataract formation. This previously unobserved ability of γS-crystallin to transfer disulfides intramolecularly suggests that it may serve as an oxidation sink for the lens after glutathione levels have become depleted during aging. γS-Crystallin thus serves as the last line of defense against oxidation in the eye lens, a result that underscores the chemical functionality of this protein, which is generally considered to play a purely structural role.

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Section: Resultsmentioning

confidence: 99%

Human γS-Crystallin–Copper Binding Helps Buffer against Aggregation Caused by Oxidative Damage

et al. 2020

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“…These signals also represent mutated proteins with wrong or disrupted disulfide bonds. The nonnative receptor forms were recovered in solution after removal of the denaturants used during purification (Fountoulakis et al, 1990).…”

Section: Resultsmentioning

confidence: 99%

“…We previously described that it is reactivated after removal of the reductants by dialysis and concluded that it contains at least one essential disulfide bond (Fountoulakis et al, 1989). On SDS-PAGE, the receptor shows a shift in mobility when reduced which also suggests that at least one of the disulfide bonds is essential for the compactness and folding of the protein (Fountoulakis et al, 1990). The extracellular ligand binding domain of the receptor includes eight cysteine residues all of which are oxidized, forming four disulfide bonds.…”

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confidence: 99%

Alignment of disulfide bonds of the extracellular domain of the interferon .gamma. receptor and investigation of their role in biological activity

Stüber¹,

Friedlein²,

Fountoulakis³

et al. 1993

Biochemistry

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The extracellular ligand binding domain of the human interferon gamma receptor includes eight cysteine residues forming four disulfide bonds. Only the nonreduced protein binds interferon gamma. We investigated the alignment of the disulfide bonds, using an enzymatically deglycosylated form of a soluble interferon gamma receptor, produced in baculovirus-infected insect cells. The soluble receptor was digested with endoproteinase Glu-C and proteinase K, and the proteolytic fragments were characterized by amino acid sequence analysis and mass spectrometry. It was found that four consecutive disulfide bonds are formed between residues Cys60-Cys68, Cys105-Cys150, Cys178-Cys183, and Cys197-Cys218. We also investigated the role of the disulfide bonds in biological activity of the receptor, using site-directed mutagenesis and by exchanging the cysteine residues for serines. The mutated proteins were expressed in Escherichia coli and analyzed for ligand binding capacity on protein blots. The assays showed that all disulfide bonds are essential for full ligand binding capacity. Double or quadruple mutations at cysteine residues 60 and 68, and residues 178, 183, 197, and 218, respectively, resulted in complete loss of the activity, whereas double mutations at residues 105 and 150, 178 and 183, and 197 and 218, respectively, resulted in a residual activity about 1 order of magnitude lower than that of the wild type. The specific antibodies gamma R38 and gamma R99 detected conformational epitopes stabilized by disulfide bonds involving cysteine residues 60 and 68, and 178 and 183, respectively.

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“…Moreover, the yields of native-like protein may be improved by further optimization. The entire extracellular IFNyR has been produced previously in E. coli, but required extraction under denaturing conditions and subsequent refolding to obtain biologically active material (Fountoulakis et al, 1990). Moreover, this E. coli derived protein showed evidence of conformational heterogeneity, and its affinity for IFNy was lower than that produced in eukaryotic cells (Gentz et al, 1992).…”

Section: Discussionmentioning

confidence: 99%

“…1988;Cofano et al, 1990;Gray et al, 1989;Hemmi et al. 1989;Kumar et al, 1989; Munro & Maniatis, 1989), and the full length soluble extracellular portion of the human IFNyR has been produced in E. coli and eukaryotic cells (Fountoulakis et al. 1990(Fountoulakis et al.…”

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confidence: 99%

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

Williams

Ruegg²,

Birch³

et al. 1995

Biochemistry

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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.

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Purification and biochemical characterization of a soluble human interferon gamma receptor expressed in Escherichia coli.

Cited by 55 publications

References 20 publications

Human γS-Crystallin–Copper Binding Helps Buffer against Aggregation Caused by Oxidative Damage

Human γS-Crystallin–Copper Binding Helps Buffer against Aggregation Caused by Oxidative Damage

Alignment of disulfide bonds of the extracellular domain of the interferon .gamma. receptor and investigation of their role in biological activity

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

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