Various authors have tested the employment of spores as particles for surface display suitable for vaccination [9–12, 19], for review see [5–8, 13]. In this work we continue the use of subunit A of the urease (UreA) of Helicobacter acinonychis, which has been used extensively as an antigen able to induce, an immune response [20–24]. In this study we were looking for improvements in spore surface display, and decided to use CotZ as the anchor protein, based on its localisation in the external layer of the outer coat of B. subtilis spores . CotZ as the anchor protein allowed us to use full-length UreA, which was not successful when CotB or CotC were chosen . This suggests good perspectives that CotZ may allow the use of large proteins for spore surface display. This ability may be connected with the preserved localization in the outer layer of the spore – with minimal disturbance of the outer layers caused by the passenger proteins. In addition, the near-surface localisation of the fusion protein should also improve surface exposure of passenger peptide/proteins, which is an important factor for successful use as an antigen. The densitometric analysis revealed that an estimated 1.4 × 102 molecules of the CotB fusion were extracted from each purified spore. In comparison to our previous construct, CotB-UreA1 (1.1 × 103) , CotZ-UreA is less abundant. Previous studies have shown that the entire UreA protein as passenger was also produced successfully when fused with CotG. However, unfortunately, the fusion was not localised on the spore surface .
The design of a fusion protein is always associated with some risk of failure in obtaining a functional construct. Some approaches are successful, but most of them are not, with the resulting proteins poorly expressed or displayed. The use of a peptide linker, which separates the components of fusions, may help to overcome some of these problems. As a first attempt, a short GGGGS linker was introduced. Unfortunately this modification resulted in loss of surface display of CotB-GGGGS-UreA protein. This might have been a result of introduction of a fragment without any secondary structure, which destabilised whole protein. We obtained promising results in experiments with the linker containing the EAAAK motif, which forms a stable alpha-helical structure . Incorporation of such a linker between fusion partners resulted in improved expression and permitted for efficient surface display of the CotB-GGGEAAAKGGG-UreA protein.
The results presented here showed that we found CotZ is potentially useful for spore surface display in addition to the previously used CotB, CotC and CotG proteins. Immunological experiment showed that CotZ-UreA fusions are the most efficient in stimulating an immunological response in comparison to other antigens in the mouse model [unpublished data].
The presence of any fusion protein may change the structure of the coat. However, the resistance properties of spores carrying proteins successfully displayed on the coat appear to be indistinguishable from the wild type ones at least in laboratory conditions tested. This gives a credit to use recombinant spores as vaccine vehicles in animal or human trials since the passage through the stomach environment should not affect their ability to stimulate immunological response.
Finally, in the case of difficulties with expression, stability or spore surface display of a given fusion we encourage others to use the -GGGEAAAKGGG- peptide linker, which may help to overcome these problems. The relatively short length of the linker makes it easy in use. It can be simply added in frame with the appropriate primer sequence used for PCR amplification of anchor or passenger protein.