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Thiols and disulfides: Difference between revisions
→Expression of proteins containing disulfides
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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 == | ||