title: Disulfide Bonds of Proteins Displayed on Spores of Bacillus subtilis Can Occur Spontaneously.
abstract: Surface display using spores of Bacillus subtilis is widely used to anchor antigens and enzymes of different sources. One open question is whether anchored proteins are able to form disulfide bonds. To answer this important question, we anchored the Escherichia coli alkaline phosphatase PhoA on the spore surface using two different surface proteins, CotB and CotZ. This enzyme needs two disulfide bonds to become active. Subsequently, we purified the spores and assayed for alkaline phosphatase activity. In both cases, we were able to recover enzymatic activity. Next, we asked whether formation of disulfide bonds occurs spontaneous or is catalyzed by thiol-disulfide oxidoreductases upon lysis of the cells. The experiment was repeated in a double-knockout mutant ΔbdbC and ΔbdbD. Since the disulfide bonds are also present on spores prepared from the double knockout, we conclude that oxidative environment after cell lysis is sufficient for disulfide formation of alkaline phosphatase.
authors.affiliation: Institute of Genetics, University of Bayreuth, 95440, Bayreuth, Germany.
authors.forename: Anne
authors.initials: A
authors.lastname: Richter
authors.forename: Wooil
authors.initials: W
authors.lastname: Kim
authors.forename: June-Hyung
authors.initials: JH
authors.lastname: Kim
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Schumann
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D001412:Bacillus subtilis / Q000378:metabolism / Q000502:physiology*; D001426:Bacterial Proteins / Q000378:metabolism*; D061505:Cell Surface Display Techniques; D004220:Disulfides / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D055786:Gene Knockout Techniques; D011490:Protein Disulfide Reductase (Glutathione) / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013171:Spores, Bacterial / Q000378:metabolism / Q000502:physiology*
chemical_list: D001426:Bacterial Proteins; D004220:Disulfides; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; C030069:spore-specific proteins, Bacillus; D011490:Protein Disulfide Reductase (Glutathione); D000469:Alkaline Phosphatase; C114089:phoA protein, E coli