IQUIBICEN   23947
INSTITUTO DE QUIMICA BIOLOGICA DE LA FACULTAD DE CIENCIAS EXACTAS Y NATURALES
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
CHARACTERIZATION OF THE CT DOMAIN OF SlpA FROM Lactobacillus acidophilus ATCC 4356 AND ITS USE AS AN ANCHOR TO DISPLAY HETEROLOGOUS PROTEINS ON THE SURFACES OF LACTIC ACID BACTERIA
Autor/es:
WAEHNER, PABLO,; FINA MARTIN, JOAQUINA; MALONE LUCÍA; ALLIEVI MARIANA C; PALOMINO MARÍA MERCEDES
Lugar:
Córdoba
Reunión:
Congreso; XI Congreso Argentino de Microbiología General; 2015
Resumen:
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 SlpA 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 means of the C-terminal (CT) region of S-layer protein SlpA from Lactobacillus acidophilus ATCC 4356.To evaluate the potential application of the CT domain of SlpA as an anchoring protein, the green fluorescent protein (GFP) was fused to the N-terminus of two different CT regions, which differ in length and quantity of charged amino acids. The fused proteins were successfully produced in Escherichia coli. Subsequently, the purified GFP-CT regions were added to various lactic acid bacteria (L. casei, L. plantarum, L. acidophilus, L.brevis and L. helveticus) cells grown in stationary phase in vitro, and the binding was viewed by fluorescence microscopy. These results indicated that the GFP could be bound to the cell surface of LAB tested under the direction of the CT region of SlpA. In addition, when cells were depleted for their native S-layer, by extraction with LiCl or SDS pretreatment exhibited an increased binding of the GFP-CT. Furthermore, L. acidophilus cells grown in a culture containing NaCl also exhibited binding to the GFP-CT.In order to evaluate which CT regions has a better binding and different conditions to improve the attachment to the Lactobacillus carrier, experiments of flow cytometry and fluorimetry are being performed. In conclusion, the CT of SlpA protein fused to a foreign protein like GFP 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 non- genetically modified organism, therefore its GRAS status is not altered.