title: Improvement of LysM-mediated surface display of designed ankyrin repeat proteins (DARPins) in recombinant and nonrecombinant strains of Lactococcus lactis and Lactobacillus Species.
abstract: Safety and probiotic properties make lactic acid bacteria (LAB) attractive hosts for surface display of heterologous proteins. Protein display on nonrecombinant microorganisms is preferred for therapeutic and food applications due to regulatory requirements. We displayed two designed ankyrin repeat proteins (DARPins), each possessing affinity for the Fc region of human IgG, on the surface of Lactococcus lactis by fusing them to the Usp45 secretion signal and to the peptidoglycan-binding C terminus of AcmA, containing lysine motif (LysM) repeats. Growth medium containing a secreted fusion protein was used to test its heterologous binding to 10 strains of species of the genus Lactobacillus, using flow cytometry, whole-cell enzyme-linked immunosorbent assay (ELISA), and fluorescence microscopy. The fusion proteins bound to the surfaces of all lactobacilli; however, binding to the majority of bacteria was only 2- to 5-fold stronger than that of the control. Lactobacillus salivarius ATCC 11741 demonstrated exceptionally strong binding (32- to 55-fold higher than that of the control) and may therefore be an attractive host for nonrecombinant surface display. Genomic comparison of the species indicated the exopolysaccharides of Lb. salivarius as a possible reason for the difference. Additionally, a 15-fold concentration-dependent increase in nonrecombinant surface display on L. lactis was demonstrated by growing bacteria with sublethal concentrations of the antibiotics chloramphenicol and erythromycin. Nonrecombinant surface display on LAB, based on LysM repeats, was optimized by selecting Lactobacillus salivarius ATCC 11741 as the optimal host and by introducing antibiotics as additives for increasing surface display on L. lactis. Additionally, effective display of DARPins on the surfaces of nonrecombinant LAB has opened up several new therapeutic possibilities.
authors.affiliation: Department of Biotechnology, Jožef Stefan Institute, Ljubljana, Slovenia Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia.
authors.forename: Petra
authors.initials: P
authors.lastname: Zadravec
authors.affiliation: Department of Biotechnology, Jožef Stefan Institute, Ljubljana, Slovenia Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia.
authors.forename: Borut
authors.initials: B
authors.lastname: Štrukelj
authors.affiliation: Department of Biotechnology, Jožef Stefan Institute, Ljubljana, Slovenia ales.berlec@ijs.si.
authors.forename: Aleš
authors.initials: A
authors.lastname: Berlec
mesh_terms: D017089:Ankyrin Repeat; D061505:Cell Surface Display Techniques / Q000379:methods*; D002473:Cell Wall / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D007778:Lactobacillus / Q000235:genetics / Q000378:metabolism*; D013294:Lactococcus lactis / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D010457:Peptidoglycan / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D010457:Peptidoglycan; D011993:Recombinant Fusion Proteins