CONICET | Buscador de Institutos y Recursos Humanos

Surface layers (S-layers) have been recognized ubiquitously in both Eubacteria and Archaea. S-layers proteins normally contain two functional regions: the self-assembly domain and the cell wall-targeting domain. Both regions have been characterized in the S-layer SA protein of Lactobacillus acidophilus ATCC 4356. The display of heterologous proteins on the cell surface of lactic acid bacteria (LAB) is an interesting and emerging area that holds great promise in the development of live vaccine delivery system. Various anchoring proteins, including S-layers, have been studied for their efficiency in attaching hybrid proteins to the cell membrane or cell wall of LAB. However, the expressed proteins were anchored to producer cells, thus making the host strain for surface display a genetically modified organism. In this study, we developed an approach for surface display of the heterologous proteins on the LAB cells by mean of S-layer protein fused to MBP (Maltose Binding Protein). For this purpose we cloned the full length S-layer of Lactobacillus acidophilus in the pMAL system fused to MBP, this fusion improved the solubility of the recombinant protein. It was shown that a substantial fraction of S-layer commonly extracted with LiCl from Lactobacillus acidophilus readily aggregates and forms a precipitate. When the fusion protein was purified, it did not show any precipitate formation. The pLC3 vector was expressed in Escherichia coli and the fusion protein was then purified by a one-step affinity purification for MBP using an amylose resin. The confirmation of the correct molecular mass of the fusion protein was checked by SDS-PAGE and Western blotting with anti MBP and anti S-layer antibodies. After overproducing the fusion protein successfully in E.coli, the purified protein was used to decorate Lactobacillus casei cells in vitro and the binding was viewed by fluorescence microscopy. In order to evaluate different conditions to improve the attachment to the Lactobacillus carrier, experiments of flow cytometry are being performed. In conclusion, the S-layer protein fused to a foreign protein like MBP was overproduced in a heterologous organism and shown to maintain its capacity to anchor to the cell surface of LAB. These surface display system offers the possibility of surface display of foreign antigens, suitable for application as an oral delivery vehicle. It is worth highlighting that the lactobacillus decorated is a nongenetically modified organism, therefore its GRAS status is not altered.