Abstract
BackgroundBacterial spores have been utilized as platforms for protein display. The best studied display systems are based on Bacillus subtilis spores in which several coat proteins have successfully been used as anchors for heterologous protein. Increasing knowledge about spore coat structure enables selection of new anchor proteins such as CotZ and CgeA. Here we describe a system of vectors for display of proteins on the surface of B. subtilis spores.ResultsWe have designed and constructed a set of 16 vectors for ectopic integration which can be used for spore surface display of heterologous proteins. There is a selection of five coat proteins: CotB, CotC, CotG, CotZ and CgeA which can be used for construction of fusions. Three of these (CotB, CotC and CotG) enable obtaining N-terminal and C-terminal fusions and other two (CotZ and CgeA) are designed to produce C-terminal fusions only. All the vectors enable introduction of an additional peptide linker between anchor and displayed protein to enhance surface display. As a selection marker trophic genes are used. Additionally we describe an example application of presented vector system to display CagA protein of Helicobacter pylori in fusion with CgeA spore coat protein.ConclusionsDescribed system of vectors is a versatile tool for display of heterologous proteins on the surface of B. subtilis spores. Such recombinant spores can be further used as for example biocatalysts or antigen-carriers in vaccine formulations. The lack of antibiotic resistance genes in the system makes such spores an interesting option for applications in which a possible release to the environment can occur.
Highlights
Bacterial spores have been utilized as platforms for protein display
The design of pCot vectors The prototype vector of the pCot set has the following properties. (i) It cannot replicate in B. subtilis but carries ColE1 replication sequences and a β-lactamase gene for amplification in E. coli. (ii) The cot/cgeA gene is cloned along with its original promoter to ensure proper expression of obtained fusion protein
It is worth stressing out that no antibiotic resistance gene selectable in B. subtilis is introduced into the vectors
Summary
Bacterial spores have been utilized as platforms for protein display. The best studied display systems are based on Bacillus subtilis spores in which several coat proteins have successfully been used as anchors for heterologous protein. The targeting and anchoring of heterologous proteins and peptides to the outer surface of bacteriophages and cells is becoming a tool for research in the fields of microbiology, molecular biology or vaccinology, as well as is utilized in the biotechnological industry. This approach is based on the idea of using naturally occurring surface proteins as an anchor for targeting proteins of interest, called passenger proteins. The central part of the spore is the core, containing partially dehydrated cytoplasm
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