Improvement of LysM-Mediated Surface Display of Designed Ankyrin Repeat Proteins (DARPins) in Recombinant and Nonrecombinant Strains of Lactococcus lactis and Lactobacillus Species

Abstract

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,...

Single plasmid systems for inducible dual protein expression and for CRISPR-Cas9/CRISPRi gene regulation in lactic acid bacterium Lactococcus lactis

“…The expression of infrared fluorescent protein (IRFP) 22 and human IgG-binding designed ankyrin repeat protein (DARPin) I07 – surface anchor fusion 23 , 24 from dual protein expression plasmids was normalized relative to their expression from pNZ8148 (pNZ-IRFP713 and pSD-I07, respectively). The absence of expression of the two model proteins from empty-plasmid backbones was confirmed.…”

“…Co-expression of proteins using pNZDual, pNZDualTT or pNZPolycist was examined using two model proteins previously used effectively in our research 24 , 27 . IRFP 22 is an infrared fluorescent protein with an emission maximum at 713 nm that has been used for in vivo imaging of bacteria in mice 27 .…”

Surface Anchoring on Lactococcus lactis by Covalent Isopeptide Bond

“…29,39 To test the feasibility of the isopeptide bond formation, we anchored one of the binding partners to the surface of L. lactis, by fusing it with Usp45 secretion signal and peptidoglycan-binding C-terminus of AcmA protein, as previously reported. 13,35 The second binding partner was fused to a reporter protein B domain that we previously applied for the assessment of surface display. 13,40 B domain fusion was isolated from E. coli or secreted from another recombinant L. lactis species (Fig.…”

“…SpyCatcher in fusion with Usp45 secretion signal 26 and the surface anchoring C-terminal domain of AcmA was displayed on the surface of L. lactis as previously reported for other proteins. 13,[35][36][37] Binding of SpyTag-B domain, isolated from E. coli, to recombinant L. lactis with surface displayed SpyCatcher was evaluated by flow cytometry using antibody recognizing B domain. Statistical-ly significant increase in MFI was observed when SpyT-B domain was incubated with L. lactis with induced Spy-Catcher expression, in comparison to control non-induced L. lactis cells, or induced L. lactis cells without the addition of SpyT-B domain (19.9%; Fig.…”

Heterologous surface display on lactic acid bacteria: non-GMO alternative?

“…salivarius ATCC 11741 has therefore been suggested as the optimal host for heterologous surface display on living bacteria. 14 Further improvement in heterologous display on living cells could also be achieved by using other non-covalent surface-binding domains ( Table 1). Significant differences in the affinity of noncovalent surface-binding domains between species have already been observed.…”