Disulfide Oxidoreductase, Recombinant (dsbA)

DsbA appears to be necessary for correct formulation of disulfide bonds in exported proteins in vivo. DsbA is useful as a standard in immunoblotting. This protein catalyses the reduction and exchange of disulfide bonds and the oxidation of free sulfhydryl groups in vitro. It is the strongest oxidant of the thioredoxin superfamily. This thio/disulfide oxidoreductase is required for efficient disulfide bond formation in the periplasm of E. coli.\n\nDsbA is a periplasmic protein isolated from E. coli having a molecular mass of 23,149 Dalton.\n\nSequence:\nThe sequence of the first five N-terminal amino acids was determined and was found to be Met-Ly-Lys-Ala-Trp.\n\nDimers and Aggregates:\n(same/less than) 1% as determined by silver-stained SDS-PAGE gel analysis.\n\nApplications:\n1. Western Blot (we recommend that the material be diluted in 1X SDS-PAGE sample buffer (1). On a 15-well minigel system, 50ng of protein per lane should be sufficient when used in a colorimetric Western Blot at a dilution of 1:10,000 as the primary antibody and an appropriate alkaline phosphatase conjugated secondary antibody for detection.)\n\nProtein Content:\nProtein quantitation was carried out by two independent methods: \n1. UV spectroscopy at 280nm. \n2. Analysis by RP-HPLC, using a calibrated solution of dsbA as a Reference Standard.\n\nEndotoxin:\n(same/less than) 0.1ng/ug (IEU/ug) of dsbA.\n\nReconstitution:\nReconstitute the lyophilized Human dsbA in sterile 18MO-cm H2O not less than 100ug/ml, which can then be further diluted to other aqueous solutions.\n\nStorage and Stability:\nLyophilized powder may be stored at 4 degrees C for short-term only. Reconstitute to nominal volume by adding sterile dH2O and store at -20 degrees C. Reconstituted product is stable for 12 months at -20 degrees C. For maximum recovery of product, centrifuge the original vial prior to removing the cap. Further dilutions can be made in assay buffer.

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SPECIFICATIONS

Catalog Number

D3778

Size

50ug

Form

Supplied as a lyophilized powder in 50mM sodium phosphate, 100mM sodium chloride.

Purity

(same/more than) 95% by RP-HPLC, FPLC, or reducing/non-reducing SDS-PAGE Silver Stain. Chromatographically purified.

References

1. Crystallization and preliminary X-ray diffraction studies of a protein disulfide oxidoreductase from Aquifex aeolicus. D'Ambrosio K, De Simone G, Rossi M, Acta Crystallogr D Biol Crystallogr 2004 Nov;60(Pt 11):2076-7 2. Functional properties of the protein disulfide oxidoreductase from the archaeon Pyrococcus furiosus: a member of a novel protein family related to protein disulfide-isomerase. Pedone E, Ren B, Rossi M, Eur J Biochem 2004 Aug;271(16):3437-48 3. Three homologues, including two membrane-bound proteins, of the disulfide oxidoreductase DsbA in Neisseria meningitidis: effects on bacterial growth and biogenesis of functional type IV pili. Tinsley CR, Voulhoux R, Tommassen J, J Biol Chem 2004 Jun 25;279(26):27078-87 4. Preferred conformations of cyclic Ac-Cys-Pro-Xaa-Cys-NHMe peptides: a model for chain reversal and active site of disulfide oxidoreductase. Park HS, Kim C, Biophys Chem 2003 Aug 1;105(1):89-104 5. Characterization of SrgA, a Salmonella enterica serovar Typhimurium virulence plasmid-encoded paralogue of the disulfide oxidoreductase DsbA, essential for biogenesis of plasmid-encoded fimbriae. Bouwman CW, Kohli M, Touchie GA, J Bacteriol 2003 Feb;185(3):991-1000 6. Structural basis for CO2 fixation by a novel member of the disulfide oxidoreductase family of enzymes, 2-ketopropyl-coenzyme M oxidoreductase/carboxylase. Nocek B, Jang SB, Clark DD, Biochemistry 2002 Oct 29;41(43):12907-13.