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. consequence -> introduce point mutations
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. Protein Engineering Obtain a protein with improved or new properties
Proteins with Novel Properties
Rational Protein Design
Nature
Random Mutagenesis

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

17. General Directed Evolution Procedure
Random mutagenesis methods

18. 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 (7x 10168) independent clones
Consequence -> not all modifications possible
-> modifications just along an evolutionary path !!!!

19. 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 !!!

20. 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 !!!

21. Used for: Elimination of neutral and deleterious mutations
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

22. 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!!!

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

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

25. 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)

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

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

28. Random Mutagenesis (PCR based)

29. Directed Evolution Difference between non-recombinative and recombinative methods
Non-recombinative methods
recombinative methods -> hybrids (chimeric proteins)

30. 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)

31. 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)

32. low activity on tributyrin
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)
colonies were screened  50 positive colonies
Name of colony
Mutations
comments
P7/7
E9K + L48L + R156R
P5/5
D21N
P5/6
D21N + R211R
P5/3
D83N
low activity on tributyrin
P5/8
D83G
P7/8
G82R + P160P
P5/2
G89G + E131K
P7/6
S4S + L15P + D132N

33. 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)

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

35. 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

36. 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)

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

38. 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 –10528, September 1998

39. 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:

40. 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:

41. 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, , 2006

42. 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:

43. 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)

44. -> w/o/w -> water in oil in water emulsion (Vesicle)
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)

45. 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.

46. Protein Engineering -> Folding (+Structure):
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 …..)

47. Protein Engineering -> Function:
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

48. 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

49. 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

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

51. Protein Engineering - Applications
Engineering Stability of Enzymes – triosephosphate isomerase from yeast
-> replace Asn (deaminated at high temperature)

52. Protein Engineering - Applications
Engineering Activity of Enzymes – tyrosyl-tRNA synthetase from B. stearothermophilus
-> replace Thr 51 (improve affinity for ATP) -> Design

53. Protein Engineering - Applications
Engineering Ca-independency of subtilisin
Saturation mutagenesis -> 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

54. 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

55. Protein Engineering – Applications Directed Evolution

56. Protein Engineering – Applications Directed Evolution

57. Protein Engineering – Applications Directed Evolution

58. Protein Engineering – Applications Directed Evolution

59. Protein Engineering – Directed Evolution

60. Protein Engineering - Applications