Figure 1From: Directed evolution and targeted mutagenesis to murinize listeria monocytogenes internalin A for enhanced infectivity in the murine oral infection modelNisin inducibe InlA plasmid constructs and the expression of InlAWT on the surface of L. lactis. A. Lactococcal nisin inducible plasmid pNZB with the entire (i) inlAWT gene from EGD-e cloned upstream of the nisin inducible nisA promoter (P). The labels for the InlA domains are described in the introduction text. The naturally occuring Bgl II/Bst XI restriction sites within the inlA gene encompass the entire LRR required for the interaction with E-cadherin. These two sites were used for the cloning of the (ii) the murinized version of inlA (inlAm*) with the amino acid changes as described by Wollert et al. [17] (created by site directed mutagenesis) and (iii) four randomly mutated banks of inlA generated by error prone PCR. B. A lysozyme cell wall extract was isolated from L. lactis InlAWT grown under the conditions used for invasion assay. Exponential phase cells (OD = 0.6) were cultured for 1 h in the presence (+) or absence (-) of nisin (approx 10 ng/ul). Proteins were run on 10% SDS-PAGE and either stained with coomassie blue or blotted and detected with a InlA monoclonal antibody [23].Back to article page