Thiols and 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.

Several possibilities are availbale for successful expression

• 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 ([1]) fusion.
• Expression in yeast [2]and secretion into the medium. Here are also several systems available. Check out the current catalogues of molecular biology supply. E.g. Pichia pastoris from Invitrogen.
• 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.

Determination of thiols and disulfides in proteins

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.

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:

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.

Method according to Thannhauser: determination of disulfides and thiols

The amount of disulfides in a protein is assessed by determination of thiols generated through cleavage of disulfides by sulfite. For the measurements a derivative of DTNB has to be prepared:

NTSB "synthesis": 29.8 mg of DTNB is dissolved in 3 ml 1 M Na₂SO₃. Adjust pH to 9-9.5. The DTNB is cleaved by the sulfite as indicated by the intense yellow color formed. The products are NTSB (2-nitro 5-thio sulfo benzoic acid) and NTB. The NTB reoxidizes with oxygen to DTNB which is subsequently cleaved again to NTSB and NTB. The progress of the conversion of DTNB into NTSB can be easily followed by decrease in 412 nm or just by the naked eye by decrease in yellow color. The residual solution is pale yellow. The final NTSB solution is 50 mM and is stable for at least 6 months at -20°C.

• 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.
• 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 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.
• 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.