Development of lactic acid bacterium capable of binding Shiga toxin on the surface

Petra Zadravec^1,2, Aleš Berlec², Petr Malý³, Borut Štrukelj^1,2

¹Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, SI-1000 Ljubljana, Slovenia
²Department of Biotechnology, Jožef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia
³Laboratory of Ligand Engineering, Institute of Biotechnology AS CR, v. v. i., Vídeňská 1083, 142 20 Prague, Czech Republic

petra.zadravec@ffa.uni-lj.si, ales.berlec@ijs.si

Lactic acid bacteria (LAB) have long history of safe usage in the dairy industry. Because of their important role in human intestine and potential health-promoting effects, LAB are considered as promising vectors for the delivery of different recombinant proteins to the human intestine. Infections with shiga toxin-producing bacteria, like enterohaemorragic Escherichia coli and Shigella dysenteriae, represent serious medical problem. Recombinant lactic acid bacteria could be engineered to bind shiga toxin on their surface by displaying binding proteins against B subunit of shiga toxin (StxB) on the surface of LAB. Small engineered binding proteins, like designed ankyrin repeat proteins (DARPins), derivatives of B domain of staphylococcal protein A (affibodies) and derivatives of albumin-binding domain (ABD) of streptococcal protein G, were reported as promising alternatives to antibodies for many applications.

In the first part of the present study, we tested the efficiency of the display of binding proteins on the surface of model LAB Lactococcus lactis. We have prepared and evaluated all three types of binding proteins (DARPins, B domain, ABD) on the surface of L. lactis.

In the second part we focused on the expression of recombinant StxB and further development of binding proteins against StxB using ABD library (1). We have prepared functional recombinant Stx1B and Stx2B as targets for selection of binding proteins. After five cycles of ribosome display and screening of positive clones with ELISA, we selected four potential Stx1B binders. Our future work will focus on further characterization of selected binders and their functional display on the surface of LAB.

1. Ahmad et al., Proteins: Struc. Funct. Bioinform. 2012, 80, 774-789.