Methods of directed evolution of PA-IIL lectin
| Authors | |
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| Year of publication | 2010 |
| Type | Conference abstract |
| MU Faculty or unit | |
| Citation | |
| Description | Directed evolution is a powerful tool widely used in protein engineering for producing proteins with altered or improved properties. This approach includes a lot of methods used to create diverse mutant libraries and to screen these libraries. In our work two mutation techniques are being used. The first one is random mutagenesis by error-prone PCR (which introduces nucleotide changes during PCR due to the use of error-prone DNA polymerase and reaction conditions). The second one is targeted-random mutagenesis (which employs mixture of special primers carried random base substitutions on selected site). LecB, the gene coding PA-IIL lectin from bacterium Pseudomonas aeruginosa, was chosen as a target gene to be modified via random mutagenesis. This soluble protein is well-characterized and it is indisputable that it displays an unusually high affinity for monosaccharides widely occurring as terminal residues in cell surface glycoconjugates highly present in cystic fibrosis mucins. The mutagenesis could help us to create a protein with improved specificity and affinity. Currently, methods for high-throughput screening of mutant libraries and their binding properties by surface plasmon resonance have been optimised to find mutants with modified affinity and specificity. The mutated lectins with improved properties could be further used in biotechnology and for bioanalytical purposes. |
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