Fig. 2

Covalent anchoring to the lactobacillal cell wall. The picture shows a schematic overview of the expression cassette for cell wall anchoring of a protein of interest, using an anchoring sequence derived from the Lp_2578 protein of L. plantarum. The cassette is translationally fused to the inducible PsppA promoter and all parts are easily interchangeable using introduced restriction sites (NdeI, SalI, MluI and multiple cloning site, MCS). The primary gene product comprises a signal peptide (indigo), which in this example is derived from Lp_2578, but which could be any peptide from a signal peptide library [23]. The predicted signal peptide cleavage site is indicated by an arrow. The protein of interest is inserted between SalI and MluI restriction sites that were engineered into the vector for this purpose (i.e. two two-residue linker sequences, in green). The protein of interest is C-terminally fuses to a C-terminal fragment of Lp_2578 including the LPxTG anchoring domain consisting of the LPxTG motif (red; the consensus sequence in L. plantarum is LPQTxE [148]), followed by a highly hydrophobic stretch (black) and positively charged C-terminal arginine residues (blue). The length of the Lp_2578 linker may be varied and so far three variants have been constructed, all of which were shown to work [41]. Full length N-terminally processed and cell-wall anchored Lp_2578 is 647 residues of which the last 194 residues are a region of low complexity. The three available linkers comprise 128, 194 and 644 residues, corresponding to a truncated low-complexity region, the complete low complexity region, and almost the complete protein, respectively