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.