title: 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.
abstract: 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 alpha-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 alpha-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.
authors.affiliation: Department of Molecular Science and Material Engineering, Graduate School of Science and Technology, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe 657-8501, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Okano
authors.forename: Qiao
authors.initials: Q
authors.lastname: Zhang
authors.forename: Sakurako
authors.initials: S
authors.lastname: Kimura
authors.forename: Junya
authors.initials: J
authors.lastname: Narita
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Tanaka
authors.forename: Hideki
authors.initials: H
authors.lastname: Fukuda
authors.forename: Akihiko
authors.initials: A
authors.lastname: Kondo
mesh_terms: D015153:Blotting, Western; D005434:Flow Cytometry; D013294:Lactococcus lactis / Q000235:genetics / Q000378:metabolism*; D050576:Membrane Fusion Proteins / Q000378:metabolism*; D009113:Muramidase / Q000235:genetics / Q000378:metabolism*; D010457:Peptidoglycan / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D020080:Tandem Repeat Sequences / Q000235:genetics
chemical_list: D050576:Membrane Fusion Proteins; D010457:Peptidoglycan; C092497:AcmA protein, Lactococcus lactis; D009113:Muramidase