Publication details

Random mutagenesis of lectins and high-throughput screening of mutant libraries

Authors

MRÁZKOVÁ Jana POKORNÁ Martina WIMMEROVÁ Michaela

Year of publication 2011
Type Conference abstract
MU Faculty or unit

Central European Institute of Technology

Citation
Description Random mutagenesis is often 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 and to screen these libraries. In our work two methodical directions are being applied. Error-prone PCR is based on error-prone DNA polymerase which introduces nucleotide changes during extension of amplified DNA. Mutational reaction conditions can support even higher number of random substitutions. Targeted-random mutagenesis is being performed by mixture of special oligonucleotide primers carried random base substitutions on selected site(s). An effective high-throughput screening must be employed while large mutant libraries are produced by random mutagenesis techniques. Currently, methods of high-throughput screening have been optimized to maximize of automation of this process. Several approaches simplifying the protein mixture for binding studies were applied. A signal sequence for expression to periplasm was added to gene of our interest and cultivation of cell culture in the presence of glycine for protein leaking out has been performed. An oligohistidine anchor was added to N terminus of proteins of our interest and chelating chromatography in mini spin columns has been tested. Due to good thermostability of target proteins, temperature clean-up of cell lysate has been optimized. LecB, the gene coding PA-IIL lectin from bacterium P. aeruginosa, was chosen as a primary target gene to be modified via 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 [1]. The aim of this work is to produce mutants with interestedly modified specificity and/or affinity for monosaccharides. The mutated lectins with improved properties could be further used in biotechnology and for bioanalytical purposes.
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