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Protein engineering

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This document discusses protein engineering techniques for modifying proteins, including rational protein design using site-directed mutagenesis and directed evolution using random mutagenesis. Site-directed mutagenesis involves introducing point mutations in a particular known area to modify a specific protein function, while directed evolution generates genetic diversity through random mutagenesis and screens variants to identify successful mutations without requiring structural information. Common random mutagenesis methods discussed are error-prone PCR and DNA shuffling, which can be used to engineer properties like protein folding, stability, binding, and catalysis.

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1 Protein Engineering
2 Protein Engineering Obtain a protein with improved or new properties Rational Protein Design Nature Proteins with Novel Properties Random Mutagenesis
3 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
4 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 5 sequence (gene) for the protein Silent mutations: -> change in nucleotide sequence with no consequences for protein sequence -> Change of amino acid -> truncation of protein -> change of c-terminal part of protein -> change of c-terminal part of protein
6 Applications of directed mutagenesis
7 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
8 Rational Protein Design Þ Site –directed mutagenesis !!! Requirements: -> Knowledge of sequence and preferable Structure (active site,….) -> Understanding of mechanism (knowledge about structure – function relationship) -> Identification of cofactors……..
9 Site-directed mutagenesis methods
10 Site-directed mutagenesis methods – Oligonucleotide - directed method
Site-directed mutagenesis methods – PCR based 11
Directed Evolution – Random mutagenesis 12 -> based on the process of natural evolution - NO structural information required - NO understanding of the mechanism required General Procedure: Generation of genetic diversity Þ Random mutagenesis Identification of successful variants Þ Screening and seletion
13
14 Random Mutagenesis (PCR based) with degenerated primers (saturation mutagenesis)
15 Random Mutagenesis (PCR based) with degenerated primers (saturation mutagenesis)
16 Random Mutagenesis (PCR based) Error –prone PCR -> PCR with low fidelity !!! Achieved by: - Increased Mg2+ concentration - Addition of Mn2+ - Not equal concentration of the four dNTPs - Use of dITP - Increasing amount of Taq polymerase (Polymerase with NO proof reading function)
17 Random Mutagenesis (PCR based) DNA Shuffling DNase I treatment (Fragmentation, 10-50 bp, Mn2+) Reassembly (PCR without primers, Extension and Recombination) PCR amplification
18 Random Mutagenesis (PCR based) Family Shuffling Genes coming from the same gene family -> highly homologous -> Family shuffling
19 Protein Engineering What can be engineered in Proteins ? -> Folding (+Structure): 1. Thermodynamic Stability (Equilibrium between: Native Û Unfolded state) 2. Thermal and Environmental Stability (Temperature, pH, Solvent, Detergents, Salt …..)
20 Protein Engineering What can be engineered in Proteins ? -> Function: 1. Binding (Interaction of a protein with its surroundings) How many points are required to bind a molecule with high affinity? 2. Catalysis (a different form of binding – binding the transition state of a chemical reaction) Increased binding to the transition state Þ increased catalytic rates !!! Requires: Knowledge of the Catalytic Mechanism !!! -> engineer Kcat and Km
21 Protein Engineering Factors which contribute to stability: 1. Hydrophobicity (hydrophobic core) 2. Electrostatic Interactions: -> Salt Bridges -> Hydrogen Bonds -> Dipole Interactions 3. Disulfide Bridges 4. Metal Binding (Metal chelating site) 5. Reduction of the unfolded state entropy with X ® Pro mutations
22 Protein Engineering Design of Thermal and Environmental stability: 1. Stabilization of a-Helix Macrodipoles 2. Engineer Structural Motifes (like Helix N-Caps) 3. Introduction of salt bridges 4. Introduction of residues with higher intrinsic properties for their conformational state (e.g. Ala replacement within a a-Helix) 5. Introduction of disulfide bridges 6. Reduction of the unfolded state entropy with X ® Pro mutations
23 Protein Engineering - Applications Engineering Stability of Enzymes – T4 lysozyme -> S-S bonds introduction
24 Protein Engineering - Applications Engineering Stability of Enzymes – triosephosphate isomerase from yeast -> replace Asn (deaminated at high temperature)
25 Protein Engineering - Applications Site-directed mutagenesis -> used to alter a single property Problem : changing one property -> disrupts another characteristics Directed Evolution (Molecular breeding) -> alteration of multiple properties
26 Protein Engineering – Applications Directed Evolution
27 Protein Engineering – Applications Directed Evolution
28 Protein Engineering – Directed Evolution
29 Protein Engineering - Applications

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Protein engineering

  • 2. 2 Protein Engineering Obtain a protein with improved or new properties Rational Protein Design Nature Proteins with Novel Properties Random Mutagenesis
  • 3. 3 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
  • 4. 4 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
  • 5. Consequences of point mutations within a coding 5 sequence (gene) for the protein Silent mutations: -> change in nucleotide sequence with no consequences for protein sequence -> Change of amino acid -> truncation of protein -> change of c-terminal part of protein -> change of c-terminal part of protein
  • 6. 6 Applications of directed mutagenesis
  • 7. 7 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
  • 8. 8 Rational Protein Design Þ Site –directed mutagenesis !!! Requirements: -> Knowledge of sequence and preferable Structure (active site,….) -> Understanding of mechanism (knowledge about structure – function relationship) -> Identification of cofactors……..
  • 10. 10 Site-directed mutagenesis methods – Oligonucleotide - directed method
  • 12. Directed Evolution – Random mutagenesis 12 -> based on the process of natural evolution - NO structural information required - NO understanding of the mechanism required General Procedure: Generation of genetic diversity Þ Random mutagenesis Identification of successful variants Þ Screening and seletion
  • 13. 13
  • 14. 14 Random Mutagenesis (PCR based) with degenerated primers (saturation mutagenesis)
  • 15. 15 Random Mutagenesis (PCR based) with degenerated primers (saturation mutagenesis)
  • 16. 16 Random Mutagenesis (PCR based) Error –prone PCR -> PCR with low fidelity !!! Achieved by: - Increased Mg2+ concentration - Addition of Mn2+ - Not equal concentration of the four dNTPs - Use of dITP - Increasing amount of Taq polymerase (Polymerase with NO proof reading function)
  • 17. 17 Random Mutagenesis (PCR based) DNA Shuffling DNase I treatment (Fragmentation, 10-50 bp, Mn2+) Reassembly (PCR without primers, Extension and Recombination) PCR amplification
  • 18. 18 Random Mutagenesis (PCR based) Family Shuffling Genes coming from the same gene family -> highly homologous -> Family shuffling
  • 19. 19 Protein Engineering What can be engineered in Proteins ? -> Folding (+Structure): 1. Thermodynamic Stability (Equilibrium between: Native Û Unfolded state) 2. Thermal and Environmental Stability (Temperature, pH, Solvent, Detergents, Salt …..)
  • 20. 20 Protein Engineering What can be engineered in Proteins ? -> Function: 1. Binding (Interaction of a protein with its surroundings) How many points are required to bind a molecule with high affinity? 2. Catalysis (a different form of binding – binding the transition state of a chemical reaction) Increased binding to the transition state Þ increased catalytic rates !!! Requires: Knowledge of the Catalytic Mechanism !!! -> engineer Kcat and Km
  • 21. 21 Protein Engineering Factors which contribute to stability: 1. Hydrophobicity (hydrophobic core) 2. Electrostatic Interactions: -> Salt Bridges -> Hydrogen Bonds -> Dipole Interactions 3. Disulfide Bridges 4. Metal Binding (Metal chelating site) 5. Reduction of the unfolded state entropy with X ® Pro mutations
  • 22. 22 Protein Engineering Design of Thermal and Environmental stability: 1. Stabilization of a-Helix Macrodipoles 2. Engineer Structural Motifes (like Helix N-Caps) 3. Introduction of salt bridges 4. Introduction of residues with higher intrinsic properties for their conformational state (e.g. Ala replacement within a a-Helix) 5. Introduction of disulfide bridges 6. Reduction of the unfolded state entropy with X ® Pro mutations
  • 23. 23 Protein Engineering - Applications Engineering Stability of Enzymes – T4 lysozyme -> S-S bonds introduction
  • 24. 24 Protein Engineering - Applications Engineering Stability of Enzymes – triosephosphate isomerase from yeast -> replace Asn (deaminated at high temperature)
  • 25. 25 Protein Engineering - Applications Site-directed mutagenesis -> used to alter a single property Problem : changing one property -> disrupts another characteristics Directed Evolution (Molecular breeding) -> alteration of multiple properties
  • 26. 26 Protein Engineering – Applications Directed Evolution
  • 27. 27 Protein Engineering – Applications Directed Evolution
  • 28. 28 Protein Engineering – Directed Evolution
  • 29. 29 Protein Engineering - Applications