Mutagenesis as a tool for improving lectins from pathogenic bacteria

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Year of publication 2012
Type Conference abstract
MU Faculty or unit

Faculty of Science

Description Directed evolution is widely used in protein engineering for producing proteins with altered or improved properties. This powerful tool includes several main approaches. The first is a random mutagenesis, which is used for creating random mutations anywhere in the gene of interest. The second is a targeted random mutagenesis, which is utilized for preparing mutants with random base substitutions in selected hotspots. In addition, a site directed mutagenesis is based on rational design and it serves for creating specific amino acid substitutions in selected hotspots. In our work two technical approaches - the targeted-random mutagenesis via mixture of oligonucleotide primers and the site directed mutagenesis via two primers carrying specific substitution – are employed. Lectins are proteins produced by different organisms. They are able to bind various sugars specifically. This fact is very important especially in host pathogen interactions where lectins help to pathogen in adhesion to sugar moieties presented on host cells. Pseudomonas aeruginosa, Ralstonia solanacearum and Burkholderia cenocepacia are opportunistic pathogens which attack mainly imunocompromised patients. These bacteria produce lectins that display significant similarities. The PA-IIL lectin from bacterium P. aeruginosa was chosen as a primary target gene to be modified via targeted random mutagenesis. This soluble protein is well-characterized and it shows very high affinity for L-fucose but it can bind several monosaccharides with similar stereochemistry e.g. L-galactose or D-mannose. Orthologues of this lectin have been identified in bacteria R. solanacearum (RS-IIL) and B. cenocepacia (BC2L-A). However, these two PA-IIL-like lectins prefer D-mannose instead of L-fucose. The aim of this work is to produce Ca-dependent lectins with a sharp specificity to L-fucose. The lectins with improved properties could be further used in biotechnology and for bioanalytical purposes.
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