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Thiols and disulfides: Difference between revisions
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Several possibilities are availbale for successful 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 ([http://www.neb.com/nebecomm/products/ | * 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 ([http://www.neb.com/nebecomm/products/productE8200.asp]) fusion. | ||
* Expression in yeast [http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Expression_systems]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 [http://www.invitrogen.com Invitrogen]. | * Expression in yeast [http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Expression_systems]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 [http://www.invitrogen.com Invitrogen]. | ||
* 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. | * 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. | ||
== Determination of thiols and disulfides in proteins == | == Determination of thiols and disulfides in proteins == | ||
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NTSB "synthesis": | NTSB "synthesis": | ||
29.8 mg of DTNB is dissolved in 3 ml 1 M Na<sub>2</sub>SO<sub>3</sub>. 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 (nitro thio | 29.8 mg of DTNB is dissolved in 3 ml 1 M Na<sub>2</sub>SO<sub>3</sub>. 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. | ||
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* 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 === | === References === | ||