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Thiols and disulfides: Difference between revisions
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* Expression targeted to the periplasm. Several vectors for ''E.coli'' are available, e.g. containing the signal sequence of OmpA or for expression as MalE fusion. | * Expression targeted to the periplasm. Several vectors for ''E.coli'' are available, e.g. containing the signal sequence of OmpA or for expression as MalE fusion. | ||
* Expression in yeast and secretion into the medium.Here are also several systems available. Check out the current catalogues of molecular biology supply. | * Expression in yeast and secretion into the medium.Here are also several systems available. Check out the current catalogues of molecular biology supply. | ||
* Expression in ''E.coli'' strains with defects in maintaining the low redox potential of the cytoplasm. Strains carrying mutations in thioredoxin reductase (trxB-, ADA494) or glutathione reductase (gor-) or both (Origami strain) allow disulfide formation in the cytoplasm. Even expression of multiple Ig domains is possible in the Origami strain. | * Expression in ''E.coli'' strains with defects in maintaining the low redox potential of the cytoplasm. Strains carrying mutations in thioredoxin reductase (trxB<sup>-</sup>, ADA494) or glutathione reductase (gor<sup>-</sup>) or both (Origami strain) allow disulfide formation in the cytoplasm. Even expression of multiple Ig domains is possible in the Origami strain. | ||
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=== Method according to Riddles: determination of thiols with DTNB === | === Method according to Riddles: determination of thiols with DTNB === | ||
Dilute protein in 1 ml of 6 M Guanidine-HCl, 50mM Tris-HCl, pH 8.3, 1 mM EDTA to a final concentration of 10-40 µM thiols. Add 10 µl of 10 mM DTNB (Dithionitrobenzoic acid; Ellman’s reagent) in 100 mM Tris-Cl, pH 7.6. The DTNB is cleaved by the thiol and a mixed disulfide of one NTB moiety with the Cys thiol is formed.The other NTB moiety has an intense absorption band at 412 nm. Extinction coefficient is 13,600 M-1 cm-1 per thiol. Make blank with buffer and DTNB, since the absorption band of DTNB tails to 412 nm. | Dilute protein in 1 ml of 6 M Guanidine-HCl, 50mM Tris-HCl, pH 8.3, 1 mM EDTA to a final concentration of 10-40 µM thiols. Add 10 µl of 10 mM DTNB (Dithionitrobenzoic acid; Ellman’s reagent) in 100 mM Tris-Cl, pH 7.6. The DTNB is cleaved by the thiol and a mixed disulfide of one NTB moiety with the Cys thiol is formed.The other NTB moiety has an intense absorption band at 412 nm. Extinction coefficient is 13,600 M<sup>-1</sup> cm<sup>-1</sup> per thiol. Make blank with buffer and DTNB, since the absorption band of DTNB tails to 412 nm. | ||
If the thiols in the protein are oxidizing very fast keep the protein at low pH, which will keep the thiol protonated. Only the thiolate is oxidizing very fast. At low pH the assay with DTNB does not work. | If the thiols in the protein are oxidizing very fast keep the protein at low pH, which will keep the thiol protonated. Only the thiolate is oxidizing very fast. At low pH the assay with DTNB does not work. | ||
=== Method according to Pedersen: determination of thiols with DTP: === | === Method according to Pedersen: determination of thiols with DTP: === | ||
Dilute protein in 950 µl of 6 M Guanidine-HCl, 100 mM acetate, pH 4.0, 1 mM EDTA to a final concentration of 10-40 µM thiols. Add 50 µl of 3.2 mM DTP (2,2-Dithiopyridin) in 0.2 M acetate, pH 4.0. Read absorption at 343 nm. Extinction coefficient is 7,600 M-1 cm-1 per thiol. | Dilute protein in 950 µl of 6 M Guanidine-HCl, 100 mM acetate, pH 4.0, 1 mM EDTA to a final concentration of 10-40 µM thiols. Add 50 µl of 3.2 mM DTP (2,2-Dithiopyridin) in 0.2 M acetate, pH 4.0. Read absorption at 343 nm. Extinction coefficient is 7,600 M<sup>-1</sup> cm<sup>-1</sup> per thiol. | ||
=== Method according to Thannhauser: determination of disulfides and thiols === | === Method according to Thannhauser: determination of disulfides and thiols === | ||