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Thiols and disulfides: Difference between revisions
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Expression of proteins containing disulfides | = Expression of proteins containing disulfides = | ||
Expression of proteins containing disulfides in the native state can make a lot of trouble when expressed using standard vectors and strains for cytoplasmic expression. | Expression of proteins containing disulfides in the native state can make a lot of trouble when expressed using standard vectors and strains for cytoplasmic expression. | ||
Several possibilities are availbale for successful expression | Several possibilities are availbale for successful expression | ||
* Expression targetetd to the periplasm | |||
* Expression in yeast and secretion into the medium | |||
* Expression in strains with defects in maintaining the low redxpotential of the cytplasm. | |||
Determination of thiols and disulfides in proteins | = Determination of thiols and disulfides in proteins = | ||
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. Read absorption at 412 nm. Extinction coefficient is 13,600 M-1 cm-1 per thiol. | 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. Read absorption at 412 nm. Extinction coefficient is 13,600 M-1 cm-1 per thiol. | ||
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1. Determine thiols as described above. | * 1. Determine thiols as described above. | ||
2. Prepare a stock of 6.3 M Guanidine-HCl, 1 mM EDTA, 0.2 M Tris-Cl, pH 9.5. | * 2. Prepare a stock of 6.3 M Guanidine-HCl, 1 mM EDTA, 0.2 M Tris-Cl, pH 9.5. | ||
3. Prepare a fresh 2 M Na2SO3 solution within 1 mM EDTA / water. | * 3. Prepare a fresh 2 M Na2SO3 solution within 1 mM EDTA / water. | ||
4. Prepare always freshly the reaction buffer by mixing 20 parts of buffer of point 2. and 1 part of | * 4. Prepare always freshly the reaction buffer by mixing 20 parts of buffer of point 2. and 1 part of 2 M Na2SO3 solution | ||
5. Dilute protein in 1ml reaction buffer at a final concentration of 10-40 µM disulfide. The disulfide is cleaved into a thiol and a thio-sulfonate. Thiols are determined by NTSB. Add 10 µl of NTSB stock as prepared above. Read absorption at 412 nm. Extinction coefficient is 13,600 M-1 cm-1 per thiol or disulfide. | * 5. Dilute protein in 1ml reaction buffer at a final concentration of 10-40 µM disulfide. The disulfide is cleaved into a thiol and a thio-sulfonate. Thiols are determined by NTSB. Add 10 µl of NTSB stock as prepared above. Read absorption at 412 nm. Extinction coefficient is 13,600 M-1 cm-1 per thiol or disulfide. | ||
6. Subtraction of number of determined thiols yields number of disulfides | * 6. Subtraction of number of determined thiols yields number of disulfides | ||
References | |||
* Pedersen, A. O., and Jacobsen, J. (1980) Reactivity of the thiol group in human and bovine albumin at pH 3-9, as measured by exchange with 2,2'-dithiodipyridine. Eur. J. Biochem. 106, 291-5. | |||
* Riddles P.W., Blakeley R.L., Zerner B. (1983) Reassessment of Ellman's reagent, Methods Enzymol. 91, 49-60. | |||
* Thannhauser TW, Konishi Y, Scheraga HA. (1987) Analysis for disulfide bonds in peptides and proteins. Methods Enzymol. 143, 115-9. | |||
Thannhauser TW, Konishi Y, Scheraga HA. Analysis for disulfide bonds in peptides and proteins. Methods Enzymol. | |||