bSafety 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. T he ability to display heterologous proteins on bacterial surfaces is becoming increasingly important in various fields of biotechnology (1-3). Bacteria displaying heterologous proteins can be used as bioadsorbents, biosensors, biocatalysts, and oral vaccines and in antibody production, screening of peptide libraries, and detection of mutations (1-3). Lactic acid bacteria (LAB) are particularly attractive hosts due to their long-term usage in food, their industrial applicability, and the general acceptance of their probiotic properties (4). Several approaches to the display of heterologous proteins on the surfaces of LAB have been established, including the use of LPXTG-type domains (5), lysine motif (LysM) domains (6), surface layer proteins (7), and lipoprotein anchors (8). Display of heterologous proteins on the surfaces of LAB has been exploited for the preparation of mucosal vaccines (9, 10), for the delivery of binding molecules to the gastrointestinal tract (11), for the assembly of macromolecular enzyme complexes (12), and for bacterial immobilization (13).The nonrecombinant approach to surface display is preferred for therapeutic and food applications. This can be achieved by noncovalent binding of recombinant proteins to the surfaces of nonrecombinant bacteria. The feasibility of such an approach has already been demonstrated by using the C-terminal part of the lactococcal AcmA protein (cA) as the cell wall anchor in lactobacilli (14) and in nonviable Gram-positive enhancer matrix (GEM) particles (15). AcmA is an autolysin (N-acetylmuramidase) with a vital role in cell division in Lactococcus lactis. It comprises an enzymatic domain at the N terminus and a peptidoglycan-binding domain at the C terminus that comprises 3 LysM repeats (14,16,17). cA has been used as a fusion partner for noncovalent attachment of heterologous proteins to the surfaces of LAB,...

Lactic acid bacteria (LAB) are food-grade hosts for surface display with potential applications in food and therapy. Alternative approaches to surface display on LAB would avoid the use of recombinant DNA technology and genetically-modified organism (GMO)-related regulatory requirements. Non-covalent surface display of proteins can be achieved by fusing them to various cell-wall binding domains, of which the Lysine motif domain (LysM) is particularly well studied. Fusion proteins have been isolated from recombinant bacteria or from their growth medium and displayed on unmodified bacteria, enabling heterologous surface display. This was demonstrated on non-viable cells devoid of protein content, termed bacteria-like particles, and on various species of genus Lactobacillus. Of the latter, Lactobacillus salivarius ATCC 11741 was recently shown to be particularly amenable for LysM-mediated display. Possible regulatory implications of heterologous surface display are discussed, particularly those relevant for the European Union.

Lactococcus lactis is a lactic acid bacterium of proven safety for use in human oral applications. For this purpose, surface display of recombinant proteins is important, and new approaches for it are being sought. Analysis of the bacterial surface proteome is essential in identifying new candidate carrier proteins for surface display. We have made two different predictions of surface-associated proteins of L. lactis MG1363 by using Augur and LocateP software, which yielded 666 and 648 proteins, respectively. Surface proteins of L. lactis NZ9000, a derivative of MG1363, were identified by using a proteomics approach. The surface proteins were cleaved from intact bacteria, and the resulting peptides were identified by mass spectrometry. The latter approach yielded 80 proteins, 34 of which were not predicted by either software. Of the 80 proteins, 7 were selected for further study. These were cloned in frame with a C-terminal hexahistidine tag and overexpressed in L. lactis NZ9000 using nisin-controlled expression. Proteins of correct molecular weight carrying a hexahistidine tag were detected. Their surface localization was confirmed with flow cytometry. Basic membrane protein A (BmpA) was exposed at the highest level. To test BmpA as a candidate carrier protein, the hexahistidine tag was replaced by the B domain of staphylococcal protein A in the genetic construct. The B domain was displayed on the surface with BmpA as a carrier. The advantage of covalent BmpA binding was demonstrated. BmpA was thus shown to be a suitable candidate for a carrier protein in lactococcal surface display.Lactic acid bacteria (LAB) are among the most intensively exploited microorganisms in the dairy industry. They are used in the fermentation of foodstuffs and as probiotics on account of their health benefits (11). Lactococcus lactis is a model LAB with industrially important applications. The genomes of several L. lactis strains have been sequenced, but strains MG1363 (33) and IL1403 (4) are most commonly used in laboratories.Various biotechnological applications of surface display of recombinant proteins in bacteria have been suggested, with vaccine delivery being the most popular in LAB (20,34). Other uses include the display of enzymes (35) as whole-cell biocatalysts, display of binding molecules such as antibodies or affibodies (35) (in diagnostics as biosensors, in therapy for pathogen or toxin removal, and in bioremediation for heavy metal binding [36]), and display of proteins for protein engineering by using combinatorial libraries and in vitro selection (8).The majority of surface display techniques have been developed for Gram-negative bacteria, with autodisplay being probably the most efficacious technique (16). However, several other approaches for surface display in Gram-positive bacteria have also been described. Transmembrane proteins, lipoproteins, LPXTG-like proteins, and cell wall-binding proteins have been displayed on the surfaces of Gram-positive bacteria (10). Two of these display types have been commonly explo...

Infections with shiga toxin-producing bacteria, like enterohemorrhagic Escherichia coli and Shigella dysenteriae, represent a serious medical problem. No specific and effective treatment is available for patients with these infections, creating a need for the development of new therapies. Recombinant lactic acid bacterium Lactococcus lactis was engineered to bind Shiga toxin by displaying novel designed albumin binding domains (ABD) against Shiga toxin 1 B subunit (Stx1B) on their surface. Functional recombinant Stx1B was produced in Escherichia coli and used as a target for selection of 17 different ABD variants (named S1B) from the ABD scaffold-derived high-complex combinatorial library in combination with a five-round ribosome display. Two most promising S1Bs (S1B22 and S1B26) were characterized into more details by ELISA, surface plasmon resonance and microscale thermophoresis. Addition of S1Bs changed the subcellular distribution of Stx1B, completely eliminating it from Golgi apparatus most likely by interfering with its retrograde transport. All ABD variants were successfully displayed on the surface of L. lactis by fusing to the Usp45 secretion signal and to the peptidoglycan-binding C terminus of AcmA. Binding of Stx1B by engineered lactococcal cells was confirmed using flow cytometry and whole cell ELISA. Lactic acid bacteria prepared in this study are potentially useful for the removal of Shiga toxin from human intestine.