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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

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Directed Mutagenesis and Protein Engineering

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  1. Directed Mutagenesis and Protein Engineering

  2. 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

  3. Consequences of point mutations within a coding 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

  4. Mutagenesis Comparison of cellular and invitro mutagenesis

  5. Applications of directed mutagenesis

  6. General strategy for directed mutagenesis • Requirements: • DNA of interest (gene or promoter) must be cloned • Expression system must be available -> for testing phenotypic change

  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. 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

  9. Rational Protein Design Nature Proteins with Novel Properties Random Mutagenesis Protein Engineering Obtain a protein with improved or new properties

  10. Rational Protein Design Site –directed mutagenesis !!! Requirements: -> Knowledge of sequence and preferable Structure (active site,….) -> Understanding of mechanism (knowledge about structure – function relationship) -> Identification of cofactors……..

  11. Site-directed mutagenesis methods Old method -> used before oligonucleotide –directed mutagenesis Limitations: -> just C-> T mutations -> randomly mutated

  12. Site-directed mutagenesis methods

  13. Site-directed mutagenesis methods – Oligonucleotide - directed method

  14. Site-directed mutagenesis methods – PCR based

  15. Directed Evolution – Random mutagenesis -> based on the process of natural evolution - NO structural information required - NO understanding of the mechanism required General Procedure: Generation of genetic diversity Random mutagenesis Identification of successful variants Screening and seletion

  16. General Directed Evolution Procedure Random mutagenesis methods

  17. Directed Evolution Library Even a large library -> (108 independent clones) will not exhaustively encode all possible single point mutations. Requirements would be: 20N independend clones -> to have all possible variations in a library (+ silent mutations) N….. number of amino acids in the protein For a small protein: -> Hen egg-white Lysozyme (129 aa; 14.6 kDa) -> library with 20129 (7x 10168) independent clones Consequence -> not all modifications possible -> modifications just along an evolutionary path !!!!

  18. Limitation of Directed Evolution • Evolutionary path must exist - > to be successful • Screening method must be available • -> You get (exactly) what you ask for!!! • -> need to be done in -> High throughput !!!

  19. Typical Directed Evolution Experiment • Successful experiments involve generally • less than 6 steps (cycles)!!! • Why? • Sequences with improved properties are rather close to the parental sequence -> along a evolutionary path • 2. Capacity of our present methods to generate novel functional sequences is rather limited -> requires huge libraries •  Point Mutations !!!

  20. 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

  21. Evolutionary MethodsType 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!!!

  22. Random Mutagenesis (PCR based) with degenerated primers (saturation mutagenesis)

  23. Random Mutagenesis (PCR based) with degenerated primers (saturation mutagenesis)

  24. 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)

  25. Random Mutagenesis (PCR based) DNA Shuffling DNase I treatment (Fragmentation, 10-50 bp, Mn2+) Reassembly (PCR without primers, Extension and Recombination) PCR amplification

  26. Random Mutagenesis (PCR based) Family Shuffling Genes coming from the same gene family -> highly homologous -> Family shuffling

  27. Random Mutagenesis (PCR based)

  28. Directed EvolutionDifference between non-recombinative and recombinative methods Non-recombinative methods recombinative methods -> hybrids (chimeric proteins)

  29. Screening: Basis for all screening & selection methods Expression Libraries ->link gene with encoded product which is responsible for enzymatic activity Kilde: Reymond, Chapter 6 (HTS screening and selection of Enzyme-encoded genes)

  30. Low-medium throughput screens -> Detection of enzymatic activity of colonies on agar plates or ”crude cell lysates” -> production of fluorophor or chromophor or halos -> Screen up to 104 colonies -> effective for isolation of enzymes with improved properties -> not so effective for isolation of variants with dramatic changes of phenotype Lipase: variants on Olive oil plates With pH indicator (brilliant green) Kilde: Reymond, Chapter 6 (HTS screening and selection of Enzyme-encoded genes)

  31. Directed Evolution on Fusarium solani pisi cutinase Screening of a random mutagenesis library of cutinase for variants with preference towards long chain fatty acid esters (Tributyrin  Olive oil) 20.000 colonies were screened  50 positive colonies

  32. HTS of enzymes with Phage Display Filamentous Pages (M13) -> Bacterial cell infected by this type of bacteriophage is constantly releasing progeny phage particles. This usually leads to a growth inhibition and a massive, continous phage production but no lysis of cells. Kilde: Reymond, Chapter 6 (HTS screening and selection of Enzyme-encoded genes)

  33. HTS of enzymes with Phage Display Kilde: Reymond, Chapter 6 (HTS screening and selection of Enzyme-encoded genes)

  34. HTS of enzymes with Phage Display -> Advantages: • Phages give a direct link between gene and protein • Display on the surface allows direct and easy access for the substrate to perform enzymatic reaction -> Challenges: - to link enzyme with product

  35. HTS of enzymes with Phage Display • Selection for catalytic antibodies with peroxidase activity. • Tyramine will be oxidized of hydrogenperoxid (peroxidase antibodies) -> produced by the phage. • Biotin-tyramine bindes irreversible with phenol-sidechanin on peroxidase antibody. • Selection via biotin-streptavidin interaction Kilde: Reymond, Chapter 6 (HTS screening and selection of Enzyme-encoded genes)

  36. HTS of enzymes with Phage Display Kilde: Reymond, Chapter 6 (HTS screening and selection of Enzyme-encoded genes)

  37. HTS of enzymes with Phage Display ”Catalytic elution” • For enzymes that are dependent on co-factors • After protein expression on the surface of the phage -> all co-factors are removed and the catalytic inactive phage/enzyme is bound to an immobilized substrate. Co-factor are added -> phage with active enzyme is eluated because substrate is converted into product. Kilder: Reymond, Chapter 6 (HTS screening and selection of Enzyme-encoded genes), H. Pedersen et al., ” A method for directed evolution and functional cloning of enzymes”, Proc. Natl. Acad. Sci. USA, Vol. 95, pp. 10523–10528, September 1998

  38. HTS of enzymes with Cell Display • Similar to phage display • Uses FRET substrate • FRET-based enzyme screening. (a) The structure of the FRET substrate: Fl, BODIPY; Q, tetramethylrhodamine. (b) Binding of FRET substrate to the cell surface of E. coli cells displaying the outer membrane protein OmpT. The positively charged FRET substrate is attached to the negatively charged polysaccharides of the cell surface. (c) Upon enzymatic cleavage of the scissile bond, the FRET substrate displays Fl fluorescence, which is otherwise quenched by Q. Kilder: Reymond, Chapter 6 (HTS screening and selection of Enzyme-encoded genes), S. Becker et al., “Ultra-high-throughput screening based on cell-surface display and fluorescence-activated cell sorting for the identification of novel biocatalysts”, Current Oppinion in Biotechnology 2004, 15:323-329

  39. HTS of enzymes with Cell Display • Yeast display of antibody scFv fragments • -> Expression of scFv on yeast cells is monitored using either the HA or c-myc epitope tags. • -> Binding of the target antigen, HIV-1 gp120, is visualized using a biotinylated mAb to a noncompetitive epitope on gp120 and fluorescent streptavidin. Gp120-binding scFvs are selected by fluorescence activated cell sorting (FACS) of the yeast cells. Kilder: Reymond, Chapter 6 (HTS screening and selection of Enzyme-encoded genes), S. Becker et al., “Ultra-high-throughput screening based on cell-surface display and fluorescence-activated cell sorting for the identification of novel biocatalysts”, Current Oppinion in Biotechnology 2004, 15:323-329

  40. In vitro compartmentalization (IVC) • Water-in-oil emulsion -> make microscopic compartments (droplet volume ~5 fL) -> no diffusion between compartments. • Each compartment contains in general one single gene and acts as an artificial cell (invitro transcription and translation) • -> Gene is linked to substrate • -> if protein is active substrate will be converted into product -> signal -> fishing out • -> direct access to gene Kilder: Reymond, Chapter 6 (HTS screening and selection of Enzyme-encoded genes), O.J. Miller, “Directed evolution by in vitro compartmentalization”, Nature Methods, vol.3, no. 7, 561-570, 2006

  41. IVC selections • Microbeads i w/o emulsion • Gene coding for a phosphotriesterase is immobilized on a microbead. • Enzyme produced with a tag (T) -> on the bead på anti-tag antibodies -> enzyme interacts with antibody -> immobilized on bead. • after translation, bead transferred to different emulsion that contains a substrate with caged-biotin • Active enzyme cleaves ester-substrate -> biotin uncaged during exposure of light -> substrate and product bind to bead (biotin-streptavidin) • Emulsions distroyed -> beads incubated with monoclonale antibodies (against product) • Incubation with secondary antibody (fluorescein-labelled) –> analysed with FACS Kilder: Reymond, Chapter 6 (HTS screening and selection of Enzyme-encoded genes), S. Becker et al., “Ultra-high-throughput screening based on cell-surface display and fluorescence-activated cell sorting for the identification of novel biocatalysts”, Current Oppinion in Biotechnology 2004, 15:323-329

  42. In vitro compartmentalization (IVC) • Advantages: • In vitro system -> allows any kind of substrate, product and chemical reaction that could be incompatible with the invivo system • Different emulsions steps -> production of enzyme is decoupled from catalytic reaction • single emulsion step -> possible to change content of compartments after translationen without distroying emulsionen Kilde: Reymond, Chapter 6 (HTS screening and selection of Enzyme-encoded genes)

  43. In vitro compartmentalization (IVC) -> w/o/w -> water in oil in water emulsion (Vesicle) Avantage: • No direct link between product and gene necessary -> keep the compartment • Can be directly analysed with FACS Kilde: Reymond, Chapter 6 (HTS screening and selection of Enzyme-encoded genes)

  44. FACS (Fluorescens-activated cell sorter) Capacity > 107 per hour • The cell suspension is entrained in the center of a narrow, rapidly flowing stream of liquid. • A vibrating mechanism causes the stream of cells to break into individual droplets. • Just before the stream breaks into droplets, the flow passes through a fluorescence measuring station where the fluorescent character of interest of each cell is measured. • An electrical charging ring is placed just at the point where the stream breaks into droplets. A charge is placed on the ring based on the immediately-prior fluorescence intensity measurement, and the opposite charge is trapped on the droplet as it breaks from the stream. The charged droplets then fall through an electrostatic deflection system that diverts droplets into containers based upon their charge. http://www.bio.davidson.edu/COURSES/GENOMICS/method/FACS.html

  45. Protein Engineering What can be engineered in Proteins ? -> Folding (+Structure): 1. Thermodynamic Stability (Equilibrium between: Native  Unfolded state) 2. Thermal and Environmental Stability (Temperature, pH, Solvent, Detergents, Salt …..)

  46. 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? • Catalysis (a different form of binding – binding the transition state of a chemical reaction) • Increased binding to the transition state  increased catalytic rates !!! • Requires: Knowledge of the Catalytic Mechanism !!! • -> engineer Kcat and Km

  47. Protein Engineering • Factors which contribute to stability: • Hydrophobicity (hydrophobic core) • Electrostatic Interactions: • -> Salt Bridges • -> Hydrogen Bonds • -> Dipole Interactions • Disulfide Bridges • Metal Binding (Metal chelating site) • Reduction of the unfolded state entropy with • X  Pro mutations

  48. Protein Engineering • Design of Thermal and Environmental stability: • Stabilization of -Helix Macrodipoles • Engineer Structural Motifes (like Helix N-Caps) • Introduction of salt bridges • Introduction of residues with higher intrinsic properties for their conformational state (e.g. Ala replacement within a -Helix) • Introduction of disulfide bridges • Reduction of the unfolded state entropy with • X  Pro mutations

  49. Protein Engineering - Applications Engineering Stability of Enzymes – T4 lysozyme -> S-S bonds introduction

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