Random mutagenesis as a tool for improving properties of PA-IIL lectin from Pseudomonas aeruginosa
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| Year of publication | 2010 |
| Type | Conference abstract |
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| Description | Random mutagenesis is the most useful approach in directed evolution, widely used in protein engineering for producing proteins with altered or improved properties. Random mutagenesis includes a lot of methods used to create diverse mutant libraries. One of these methods is error-prone PCR. This technique introduces nucleotide changes during PCR due to the use of error-prone DNA polymerase and mutational reaction conditions. LecB is the target gene we have modified via random mutagenesis. This gene codes PA-IIL lectin from Pseudomonas aeruginosa. This lectin 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 random mutagenesis could help us to create a protein with interestingly modified properties towards monospecificity. After several trials with commercial screens that haven’t brought expected percentage of mutated amino acids, we have optimized error-prone reaction conditions to give sufficient results. Finally, we obtained an efficient method which introduces non-silent random mutations into the gene of our interest in sufficient rate. Currently, methods for high throughput screening of mutant libraries 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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