Directed Mutagenesis and Protein Engineering

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Directed Mutagenesis and Protein Engineering
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Mutagenesis

• Mutagenesis -gt change in DNA sequence
• -gt Point mutations or large modifications
• Point mutations (directed mutagenesis)
• Substitution change of one nucleotide (i.e. A-gt
  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
Silent mutations -gt change in nucleotide
sequence with no consequences for protein
sequence
-gt Change of amino acid
-gt truncation of protein
-gt change of c-terminal part of protein
-gt change of c-terminal part of protein
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Mutagenesis Comparison of cellular and invitro
mutagenesis
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Applications of directed mutagenesis
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General strategy for directed mutagenesis

• Requirements
• DNA of interest (gene or promoter) must be
  cloned
• Expression system must be available -gt for
  testing phenotypic change

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Approaches for directed mutagenesis

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

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Protein Engineering

• -gt Mutagenesis used for modifying proteins
• Replacements on protein level -gt 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 -gt introduce point mutations

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Protein Engineering Obtain a protein with
improved or new properties
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Rational Protein Design
? Site directed mutagenesis !!!
Requirements -gt Knowledge of sequence and
preferable Structure (active site,.) -gt
Understanding of mechanism (knowledge about
structure function relationship) -gt
Identification of cofactors..
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Site-directed mutagenesis methods
Old method -gt used before oligonucleotide
directed mutagenesis Limitations -gt just C-gt
T mutations -gt randomly mutated
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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
-gt 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
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General Directed Evolution Procedure
Random mutagenesis methods
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Directed Evolution Library
Even a large library -gt (108 independent clones)
will not exhaustively encode all possible single
point mutations. Requirements would be 20N
independend clones -gt to have all possible
variations in a library ( silent
mutations) N.. number of amino acids in the
protein For a small protein -gt Hen
egg-white Lysozyme (129 aa 14.6 kDa)
-gt library with 20129 (7x
10168) independent clones Consequence -gt not all
modifications possible -gt
modifications just along an evolutionary path
!!!!
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Limitation of Directed Evolution

• Evolutionary path must exist - gt to be successful
  
• Screening method must be available
• -gt You get (exactly) what you ask for!!!
• -gt need to be done in -gt High throughput
  !!!

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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 -gt along a
  evolutionary path
• 2. Capacity of our present methods to generate
  novel functional sequences is rather limited -gt
  requires huge libraries
• ? Point Mutations !!!

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Evolutionary Methods

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

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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
• -gt improve fitness of mutants!!!

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Random Mutagenesis (PCR based) with degenerated
primers (saturation mutagenesis)
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Random Mutagenesis (PCR based) with degenerated
primers (saturation mutagenesis)
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Random Mutagenesis (PCR based) Error prone PCR
-gt 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)
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Random Mutagenesis (PCR based) DNA Shuffling
DNase I treatment (Fragmentation, 10-50 bp, Mn2)
Reassembly (PCR without primers, Extension and
Recombination)
PCR amplification
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Random Mutagenesis (PCR based) Family Shuffling
Genes coming from the same gene family -gt highly
homologous -gt Family shuffling
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Random Mutagenesis (PCR based)
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Directed EvolutionDifference between
non-recombinative and recombinative methods
Non-recombinative methods
recombinative methods -gt hybrids (chimeric
proteins)
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Screening Basis for all screening selection
methods

• Expression Libraries
• -gtlink gene with encoded product which is
  responsible for enzymatic activity

Kilde Reymond, Chapter 6 (HTS screening and
selection of Enzyme-encoded genes)
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Low-medium throughput screens

• -gt Detection of enzymatic activity of colonies on
  agar plates or crude cell lysates -gt production
  of fluorophor or chromophor or halos
• -gt Screen up to 104 colonies
• -gt effective for isolation of enzymes with
  improved properties
• -gt 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)
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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
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HTS of enzymes with Phage Display
Filamentous Pages (M13) -gt 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)
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HTS of enzymes with Phage Display
Kilde Reymond, Chapter 6 (HTS screening and
selection of Enzyme-encoded genes)
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HTS of enzymes with Phage Display

• -gt 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
• -gt Challenges
• - to link enzyme with product

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HTS of enzymes with Phage Display

• Selection for catalytic antibodies with
  peroxidase activity.
• Tyramine will be oxidized of hydrogenperoxid
  (peroxidase antibodies) -gt 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)
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HTS of enzymes with Phage Display
Kilde Reymond, Chapter 6 (HTS screening and
selection of Enzyme-encoded genes)
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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 -gt all co-factors are removed and the
  catalytic inactive phage/enzyme is bound to an
  immobilized substrate. Co-factor are added -gt
  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. 1052310528, September 1998
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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, 15323-329
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HTS of enzymes with Cell Display

• Yeast display of antibody scFv fragments
• -gt Expression of scFv on yeast cells is
  monitored using either the HA or c-myc epitope
  tags.
• -gt 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, 15323-329
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In vitro compartmentalization (IVC)

• Water-in-oil emulsion -gt make microscopic
  compartments (droplet volume 5 fL) -gt no
  diffusion between compartments.
• Each compartment contains in general one single
  gene and acts as an artificial cell (invitro
  transcription and translation)
• -gt Gene is linked to substrate
• -gt if protein is active substrate will be
  converted into product -gt signal -gt fishing out
• -gt 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
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IVC selections

• Microbeads i w/o emulsion
• Gene coding for a phosphotriesterase is
  immobilized on a microbead.
• Enzyme produced with a tag (T) -gt on the bead på
  anti-tag antibodies -gt enzyme interacts with
  antibody -gt immobilized on bead.
• after translation, bead transferred to different
  emulsion that contains a substrate with
  caged-biotin
• Active enzyme cleaves ester-substrate -gt biotin
  uncaged during exposure of light -gt substrate
  and product bind to bead (biotin-streptavidin)
• Emulsions distroyed -gt beads incubated with
  monoclonale antibodies (against product)
• Incubation with secondary antibody
  (fluorescein-labelled) gt 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, 15323-329
43
In vitro compartmentalization (IVC)

• Advantages
• In vitro system -gt allows any kind of substrate,
  product and chemical reaction that could be
  incompatible with the invivo system
• Different emulsions steps -gt production of
  enzyme is decoupled from catalytic reaction
• single emulsion step -gt possible to change
  content of compartments after translationen
  without distroying emulsionen

Kilde Reymond, Chapter 6 (HTS screening and
selection of Enzyme-encoded genes)
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-gt w/o/w -gt water in oil in water emulsion
(Vesicle)
In vitro compartmentalization (IVC)

• Avantage
• No direct link between product and gene necessary
  -gt keep the compartment
• Can be directly analysed with FACS

Kilde Reymond, Chapter 6 (HTS screening and
selection of Enzyme-encoded genes)
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FACS (Fluorescens-activated cell sorter)
Capacity gt 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/metho
d/FACS.html
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Protein Engineering
What can be engineered in Proteins ? -gt Folding
(Structure) 1. Thermodynamic Stability
(Equilibrium between Native ? Unfolded
state) 2. Thermal and Environmental Stability
(Temperature, pH, Solvent, Detergents, Salt
..)
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Protein Engineering

• What can be engineered in Proteins ?
• -gt 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
  !!!
• -gt engineer Kcat and Km

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Protein Engineering

• Factors which contribute to stability
• Hydrophobicity (hydrophobic core)
• Electrostatic Interactions
• 
  -gt Salt Bridges
• 
  -gt Hydrogen Bonds
• 
  -gt Dipole Interactions
• Disulfide Bridges
• Metal Binding (Metal chelating site)
• Reduction of the unfolded state entropy with
• X ? Pro mutations

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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

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Protein Engineering - Applications
Engineering Stability of Enzymes T4 lysozyme
-gt S-S bonds introduction
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Protein Engineering - Applications
Engineering Stability of Enzymes
triosephosphate isomerase from yeast
-gt replace Asn (deaminated at high temperature)
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Protein Engineering - Applications
Engineering Activity of Enzymes tyrosyl-tRNA
synthetase from B. stearothermophilus
-gt replace Thr 51 (improve affinity for ATP) -gt
Design
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Protein Engineering - Applications
Engineering Ca-independency of subtilisin
Saturation mutagenesis -gt 7 out of 10 regions
were found to give increase of stability Mutant
10x more stable than native enzyme in absence of
Ca 50 more stable than native in presence of Ca
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Protein Engineering - Applications
Site-directed mutagenesis -gt used to alter a
single property Problem changing one property
-gt disrupts another characteristics Directed
Evolution (Molecular breeding) -gt alteration of
multiple properties
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Protein Engineering ApplicationsDirected
Evolution
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Protein Engineering ApplicationsDirected
Evolution
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Protein Engineering ApplicationsDirected
Evolution
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Protein Engineering ApplicationsDirected
Evolution
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Protein Engineering Directed Evolution
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Protein Engineering - Applications
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