Development of Bacillus subtilis spores and cells for surface display of proteins
Surface display has attracted the attention of researchers in developing efficient display systems expressing heterologous polypeptides on the surface of bioparticles such as phages, bacterial and eukaryotic cells and bacterial spores. Among these bioparticles, the endospore from B. subtilis has advantages, including feasibility of production, safety feature, the robustness of the bacterial spore allowing storage in the desiccated form, a technological platform supported by extensive tools for genetic manipulation and less size restrictions of the displayed proteins compared to cell- and phage-based systems. A strategy to engineer B. subtilis spores to display heterologous protein on their surface is to use outer spore coat proteins (CotB, CotC, CotG) or an inner-coat protein (OxdD) with the coat genes’ transcriptional and translational signals as carriers (Isticato et al., 2001; Mauriello et al., 2004; Hinc et al., 2010; Zhou et al., 2008a; Potot et al., 2010; Kim et al., 2005a; Kwon et al., 2007). This strategy guarantees the timing for fusion protein synthesis during coat formation, but the amount of produced fusion proteins cannot be controlled. Therefore, the first aim of this doctoral thesis focused on construction of more effective expression systems for spore surface protein anchoring. A novel approach of substitution of native promoter by two different IPTG-inducible promoters to the increase the production of fusion protein is presented here. CotB was used and the expression of the cotB gene was regulated by either its own promoter, the Pgrac and the PSgrac promoter in a series of plasmids which can be integrated into or replicated independently of the B. subtilis chromosomal DNA. Two reporter proteins, α-amylase Q from B. amyloliquefaciens (AmyQ) (Palva, 1982) and GFPuv – an enhanced version from the GFP protein of the jellyfish Aequorea victoria (Crameri et al., 1996), were fused downstream of the CotB protein. To assess the enhancement of GFPuv displayed on the spore surface, CotC and CotG were similarly examined. The results indicated that the Pgrac promoter is a suitable, hence recommended as a promoter of choice. Substitution of the native promoter by Pgrac promoter, the amount of proteins displayed per spore can be increased two-fold. Furthermore, the display of heterologous proteins on the spore surface when using different carriers is gene dosage dependent. And for the first time, the tendency of the three Cot proteins’ localization on the spore coat compartment is reported using the GFPuv tag. Second, a new B. subtilis spore-based system for protein expression and purification was developed. Using this system, proteins prone to form inclusion bodies can be anchored on the spore surface, separated by a mini-intein derived from the SSp DnaB, which was then used as self-cleaving tag for purification by shifting the pH and/or temperature conditions, with no addition of any proteases or thiol reagent (Mathys et al., 1999). To construct the system, the mini-intein was fused downstream of the CotB protein, followed by the reporter protein AmyQ. By changing the pH of the buffer, the mini-intein self-cleaving process was induced followed by the release of α-amylase into the supernatants. This observation suggests the use of the B. subtilis spores as an effective and low cost tool for protein purification. However, concerns related to premature of the pH-inducible mini-intein and auto-release of coat protein raise the question about the stability of the fusion coat-heterologous protein on the spore surface using the system. Hence, further investigation is needed to achieve a usable spore-based purification system. The last aim of the thesis was to apply the newly constructed B. subtilis spore display and the cell surface display systems (Nguyen and Schumann, 2006) to generate cellulose chips, in which enzymes were immobilized on the surface of microorganism cells or spores. The cellulase A (CelA) from C. thermocellum (Beguin et al., 1985) was utilized as a model enzyme. Unfortunately, the results showed an ineffective anchoring of CelA on the cell wall. This indicates the unsuccessful creation of cell-based cellulase chip when using the SrtA transpeptidase. In contrast, CelA was verified to be successfully displayed on the spore surface using CotB and CotG, but not CotC, as carriers. In general, a large volume of culture (up to one liter) must be prepared containing both cells and spores displaying CelA on the surface to assure sufficient CMC degradation. This might indicate a low activity of CelA. Further works should be done in selection of cellulase and improvement of the systems to generate the more effective cellulase chips.
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