System Using Tandem Repeats of the cA Peptidoglycan-Binding Domain from Lactococcus lactis for Display of both N- and C-Terminal Fusions on Cell Surfaces of Lactic Acid Bacteria

Department of Molecular Science and Material Engineering, Graduate School of Science and Technology, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe 657-8501, Japan.
Applied and Environmental Microbiology (Impact Factor: 3.67). 03/2008; 74(4):1117-23. DOI: 10.1128/AEM.02012-07
Source: PubMed


Here, we established a system for displaying heterologous protein to the C terminus of the peptidoglycan-binding domain (cA
domain) of AcmA (a major autolysin from Lactococcus lactis). Western blot and flow cytometric analyses revealed that the fusion proteins (cA-AmyA) of the cA domain and α-amylase from
Streptococcus bovis 148 (AmyA) are efficiently expressed and successfully displayed on the surfaces of L. lactis cells. AmyA was also displayed on the cell surface while retaining its activity. Moreover, with an increase in the number
of cA domains, the quantity of cA-AmyA fusion proteins displayed on the cell surface increased. When three repeats of the
cA domain were used as an anchor protein, 82% of α-amylase activity was detected on the cells. The raw starch-degrading activity
of AmyA was significantly higher when AmyA was fused to the C terminus of the cA domain than when it was fused to the N terminus.
In addition, cA-AmyA fusion proteins were successfully displayed on the cell surfaces of Lactobacillus plantarum and Lactobacillus casei.

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    • "Surface-anchored complexes were thus displayed with efficiencies that could approach 10 4 complexes/cell (Wieczorek and Martin, 2010) (Table 1). A non-covalent surface display system for LAB has also been developed by fusing a target heterologous protein, i.e., the α-amylase, with the C-terminal cA peptidoglycan-binding domain, which shows high homology with LysM repeats of the major autolysin AcmA from Lc. lactis (Okano et al., 2008). "
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    • "Degradation products of the fusion proteins were observed, most probably as a consequence of proteolytic activities in the various bacterial species. Western hybridization and flow cytometry analyses in L. lactis IL1403 revealed that an increase in the number of LysM domains leads to an increase in the amount of cell wall-located fusion protein (Okano et al. 2008). A similar observation was made using an N-terminal fusion to GFP of the LysM domain of the glucosaminidase Mbg of B. thuringiensis, which is located at the N-terminal of this enzyme (Shao et al. 2012). "
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    • "For instance, LysM domains are currently employed in the development of pneumococcal and influenza vaccines (Audouy et al. 2006, 2007; Bosma et al. 2006; Saluja et al. 2010). They are also used for the display of various heterologous proteins on the bacterial cell surface (Steen et al. 2003; Bosma et al. 2006; Okano et al. 2008; Tarahomjoo et al. 2008; Hu et al. 2010). In bacteria, LysM domains are present in murein hydrolases helping in the non-covalent attachment of the enzymes to the murein layer, thus facilitating substrate hydrolysis. "
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