Figure 7 C . When oxidized in a glutathione redox buffer, E5 forms disulfide-linked dimers, which can be observed in nonreducing SDS-PAGE. M, marker, MW in kDa indicated on the right, ox. and red.: fully oxidized and reduced E5, respectively. D , the redox potential can be calculated from the ratio of reduced and oxidized protein in a given glutathione redox buffer based on the Nernst equation. Based on these calculations, E5 has a standard redox potential of −246 mV, close to the redox potential of thioredoxin. E , reduced E5 and E. coli TrxA reduce insulin when no DTT for enzyme recycling is added. F , E5 protein oxidized in this reaction forms disulfide-linked dimers, which can be observed in a Coomassie-stained SDS-PAGE. Higher molecular weight species indicated by arrows are presumably insulin adducts to the E5 dimer and E5 multimers crosslinked by insulin. G , an E5 C77S mutant, which contains only one cysteine, still forms a disulfide-linked dimer when oxidized with diamide and ( H ) is still active in an insulin reduction assay, while an E5 C74S mutant is inactive. I , size exclusion choromatography of E5 and its C77S mutant suggests a molecular weight consistent approximately with a homohexamer (the calculated mass of E5 is 23,580 Da), when compared with a protein mixture used as standard (THY, thyroglobulin; FER, ferritin; ALD, aldolase; CON, conalbumin; OVA, ovalbumin; CAR, carbonic anhydrase; and RIB, ribonuclease). To calculate the molecular weight of E5, high and low molecular weight standard proteins were run in separate runs, and their molecular mass was plotted against the elution volume. The plot was fitted using an exponential function in Excel. " width="100%" height="100%">
Journal: The Journal of Biological Chemistry
Article Title: Functional metagenomics of the thioredoxin superfamily
doi: 10.1074/jbc.RA120.016350
Figure Lengend Snippet: Metagenomic thioredoxin E5 is a new i thioredoxin superfamily member with an unusual reaction mechanism. A , E5 is an effective disulfide isomerase in the commercial Escherichia coli SHUFFLE strain and can support oxidative folding of several proteins, including vtPA, which contains nine disulfide bonds when folded correctly. B , it shows higher initial velocity than E. coli TrxA in an insulin reduction assay. C , redox potential determination of E5 using 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid, as described for Figure 7 C . When oxidized in a glutathione redox buffer, E5 forms disulfide-linked dimers, which can be observed in nonreducing SDS-PAGE. M, marker, MW in kDa indicated on the right, ox. and red.: fully oxidized and reduced E5, respectively. D , the redox potential can be calculated from the ratio of reduced and oxidized protein in a given glutathione redox buffer based on the Nernst equation. Based on these calculations, E5 has a standard redox potential of −246 mV, close to the redox potential of thioredoxin. E , reduced E5 and E. coli TrxA reduce insulin when no DTT for enzyme recycling is added. F , E5 protein oxidized in this reaction forms disulfide-linked dimers, which can be observed in a Coomassie-stained SDS-PAGE. Higher molecular weight species indicated by arrows are presumably insulin adducts to the E5 dimer and E5 multimers crosslinked by insulin. G , an E5 C77S mutant, which contains only one cysteine, still forms a disulfide-linked dimer when oxidized with diamide and ( H ) is still active in an insulin reduction assay, while an E5 C74S mutant is inactive. I , size exclusion choromatography of E5 and its C77S mutant suggests a molecular weight consistent approximately with a homohexamer (the calculated mass of E5 is 23,580 Da), when compared with a protein mixture used as standard (THY, thyroglobulin; FER, ferritin; ALD, aldolase; CON, conalbumin; OVA, ovalbumin; CAR, carbonic anhydrase; and RIB, ribonuclease). To calculate the molecular weight of E5, high and low molecular weight standard proteins were run in separate runs, and their molecular mass was plotted against the elution volume. The plot was fitted using an exponential function in Excel.
Article Snippet: Then, samples were incubated for 5 min at 95 °C in 1× nonreducing SDS loading buffer (2% [w/v] SDS, 12% [v/v] glycerol, 60 mM Tris–HCl [pH 7.0], and 0.0001% [w/v] bromophenol blue) and loaded onto a precast NuPAGE Novex 4 to 12% Bis–Tris protein gel (Life Technologies).
Techniques: Insulin Reduction Assay, SDS Page, Marker, Enzyme Recycling Assay, Staining, Molecular Weight, Mutagenesis
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