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Слайды и текст этой презентации

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Directed Mutagenesis and Protein Engineering

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Mutagenesis Mutagenesis -> change in DNA sequence -> Point mutations or large modifications Point mutations (directed mutagenesis): Substitution: change of one nucleotide (i.e. A-> C) Insertion: gaining one additional nucleotide Deletion: loss of one nucleotide

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Consequences of point mutations within a coding sequence (gene) for the protein

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Mutagenesis Comparison of cellular and invitro mutagenesis

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General strategy for directed mutagenesis

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Approaches for directed mutagenesis -> site-directed mutagenesis -> point mutations in particular known area result -> library of wild-type and mutated DNA (site-specific) not really a library -> just 2 species -> random mutagenesis -> point mutations in all areas within DNA of interest result -> library of wild-type and mutated DNA (random) a real library -> many variants -> screening !!! if methods efficient -> mostly mutated DNA

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Protein Engineering -> Mutagenesis used for modifying proteins Replacements on protein level -> mutations on DNA level Assumption : Natural sequence can be modified to improve a certain function of protein This implies: Protein is NOT at an optimum for that function Sequence changes without disruption of the structure (otherwise it would not fold) New sequence is not TOO different from the native sequence (otherwise loss in function of protein) consequence -> introduce point mutations

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Protein Engineering Obtain a protein with improved or new properties

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Rational Protein Design

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Site-directed mutagenesis methods

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Site-directed mutagenesis methods

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Site-directed mutagenesis methods – Oligonucleotide - directed method

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Site-directed mutagenesis methods – PCR based

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Directed Evolution – Random mutagenesis

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General Directed Evolution Procedure

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Directed Evolution Library

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Limitation of Directed Evolution

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Typical Directed Evolution Experiment

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Evolutionary Methods Non-recombinative methods: -> Oligonucleotide Directed Mutagenesis (saturation mutagenesis) -> Chemical Mutagenesis, Bacterial Mutator Strains -> Error-prone PCR Recombinative methods -> Mimic nature’s recombination strategy Used for: Elimination of neutral and deleterious mutations -> DNA shuffling -> Invivo Recombination (Yeast) -> Random priming recombination, Staggered extention precess (StEP) -> ITCHY

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Evolutionary Methods Type of mutation – Fitness of mutants Type of mutations: Beneficial mutations (good) Neutral mutations Deleterious mutations (bad) Beneficial mutations are diluted with neutral and deleterious ones !!! Keep the number of mutations low per cycle -> improve fitness of mutants!!!