Table 1.
Plasmids used in this study.
Figure 1.
Single expression cassettes for surface display of ARP3 fused to VSV-G (A); and V5/HA/FLAG-tag (B); and co-expression cassettes where one ARP fragment can be secreted (ARP1) and the other (ARP3) anchored on the cell surface (C); both covalently anchored on the cell surface (D); or both secreted in the medium (E).
SP, signal peptide; RBS, ribosomal binding site; P, apf promoter; translational stop codon indicated with an arrowhead and the transcription terminator indicated with a lollipop.
Figure 2.
Expression of ARP1 and ARP3 by transformed L. paracasei BL23 in Western blot.
Expression of surface-anchored (A) and secreted (B) ARP1, and surface-anchored (C) and secreted (D) ARP3 by L. paracasei BL23 transformed with pAF100, pAF900, pAF1200, pAF1300 and pAF1400 vectors was analyzed in supernatant and cell extracts. S, supernatant; C, cell extract.
Table 2.
Amount of ARP1 and ARP3 in transformed Lactobacillus BL23 producing one or two ARPs.
Figure 3.
Surface display of ARP1 and ARP3 by transformed L. paracasei BL23 in flow cytometry.
Surface display of anchored ARP1 (A) and ARP3 (B) produced by L. paracasei BL23 transformed with vectors mediating single and co-expression of ARPs as compared to non-transformed wild-type L. paracasei BL23.
Figure 4.
Binding activity of anchored ARP1 and ARP3 produced by transformed L. paracasei BL23 cells to human and simian rotavirus strains with distinct genotypes in flow cytometry.
Figure 5.
Binding activity of culture supernatant from transformed L. paracasei BL23 producing secreted ARP1 and ARP3 to human and simian rotavirus strains with distinct genotypes in ELISA.
The binding of secreted ARP1 (A) and ARP3 (B) produced in the supernatant of L. paracasei BL23 transformed with pAF100, pAF1200 and pAF1300 vectors was compared to non-transformed wild-type L. paracasei BL23.
Figure 6.
Simultaneous binding experiment: A binding scenario in situ.
Following incubation of L. paracasei pAF1300 with rotavirus (RRV) in MRS medium for 30 min and 1 hour, RRV bound on the surface of modified lactobacilli was detected using biotinylated HBC anti-rotavirus antibodies and PE conjugated streptavidin, and secreted ARP1 bound on the surface of rotavirus was detected using a mouse monoclonal anti-E tag antibody, and FITC conjugated anti-mouse antibodies. The double-headed arrow indicates the positive shift in fluorescence intensity.