Презентация на тему: Directed Mutagenesis and Protein Engineering
Directed Mutagenesis and Protein Engineering
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
Consequences of point mutations within a coding sequence (gene) for the protein
Mutagenesis Comparison of cellular and invitro mutagenesis
General strategy for directed mutagenesis
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
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
Protein Engineering Obtain a protein with improved or new properties
Rational Protein Design
Site-directed mutagenesis methods
Site-directed mutagenesis methods
Site-directed mutagenesis methods – Oligonucleotide - directed method
Site-directed mutagenesis methods – PCR based
Directed Evolution – Random mutagenesis
General Directed Evolution Procedure
Directed Evolution Library
Limitation of Directed Evolution
Typical Directed Evolution Experiment
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
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!!!
Random Mutagenesis (PCR based) with degenerated primers (saturation mutagenesis)
Random Mutagenesis (PCR based) with degenerated primers (saturation mutagenesis)
Random Mutagenesis (PCR based) Error –prone PCR
Random Mutagenesis (PCR based) DNA Shuffling
Random Mutagenesis (PCR based) Family Shuffling
Random Mutagenesis (PCR based)
Directed Evolution Difference between non-recombinative and recombinative methods
Protein Engineering
Protein Engineering
Protein Engineering
Protein Engineering
Protein Engineering - Applications
Protein Engineering - Applications
Protein Engineering - Applications
Protein Engineering - Applications
Protein Engineering - Applications
Protein Engineering – Applications Directed Evolution
Protein Engineering – Applications Directed Evolution
Protein Engineering – Applications Directed Evolution
Protein Engineering – Applications Directed Evolution
Protein Engineering – Directed Evolution
Protein Engineering - Applications
