Directed Mutagenesis and Protein Engineering

Сентябрь 1, 2022

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Directed Mutagenesis and Protein Engineering

Содержание

2. Mutagenesis Mutagenesis -> change in DNA sequence -> Point mutations or large modifications Point mutations (directed
3. Consequences of point mutations within a coding sequence (gene) for the protein Silent mutations: -> change
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
7. Approaches for directed mutagenesis -> site-directed mutagenesis -> point mutations in particular known area result ->
8. Protein Engineering -> Mutagenesis used for modifying proteins Replacements on protein level -> mutations on DNA
9. Protein Engineering Obtain a protein with improved or new properties
10. Rational Protein Design ⇒ Site –directed mutagenesis !!! Requirements: -> Knowledge of sequence and preferable Structure
11. Site-directed mutagenesis methods Old method -> used before oligonucleotide –directed mutagenesis Limitations: -> just C-> T
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
17. General Directed Evolution Procedure Random mutagenesis methods
18. Directed Evolution Library Even a large library -> (108 independent clones) will not exhaustively encode all
19. Limitation of Directed Evolution Evolutionary path must exist - > to be successful Screening method must
20. Successful experiments involve generally less than 6 steps (cycles)!!! Why? Sequences with improved properties are rather
21. Evolutionary Methods Non-recombinative methods: -> Oligonucleotide Directed Mutagenesis (saturation mutagenesis) -> Chemical Mutagenesis, Bacterial Mutator Strains
22. Evolutionary Methods Type of mutation – Fitness of mutants Type of mutations: Beneficial mutations (good) Neutral
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: -
26. Random Mutagenesis (PCR based) DNA Shuffling DNase I treatment (Fragmentation, 10-50 bp, Mn2+) Reassembly (PCR without
27. Random Mutagenesis (PCR based) Family Shuffling Genes coming from the same gene family -> highly homologous
28. Random Mutagenesis (PCR based)
29. Directed Evolution Difference between non-recombinative and recombinative methods Non-recombinative methods recombinative methods -> hybrids (chimeric proteins)
30. Protein Engineering What can be engineered in Proteins ? -> Folding (+Structure): 1. Thermodynamic Stability (Equilibrium
31. Protein Engineering What can be engineered in Proteins ? -> Function: 1. Binding (Interaction of a
32. Protein Engineering Factors which contribute to stability: Hydrophobicity (hydrophobic core) Electrostatic Interactions: -> Salt Bridges ->
33. Protein Engineering Design of Thermal and Environmental stability: Stabilization of α-Helix Macrodipoles Engineer Structural Motifes (like
34. Protein Engineering - Applications Engineering Stability of Enzymes – T4 lysozyme -> S-S bonds introduction
35. Protein Engineering - Applications Engineering Stability of Enzymes – triosephosphate isomerase from yeast -> replace Asn
36. Protein Engineering - Applications Engineering Activity of Enzymes – tyrosyl-tRNA synthetase from B. stearothermophilus -> replace
37. Protein Engineering - Applications Engineering Ca-independency of subtilisin Saturation mutagenesis -> 7 out of 10 regions
38. DNA shuffling JCohen. News note: How DNA shuffling works. Sci 293:237 (2001) Maxygen, PCR without synthetic
39. Altering multiple properties: rapid high-throughput screening ex., subtilisin Use 26 different subtilisin genes Shuffle DNA, construct
40. Laundry, detergent and mushrooms Peroxidase, ink cap mushroom; dye transfer inhibitor Wash conditions: bleach-containing detergents, pH
41. ex., Coprinus cinereus heme peroxidase (ink cap mushroom); 343 AAc, heme prosthetic group Multiple rounds of
42. Molecular analysis of hybrid peroxidase
43. Decreasing protein sensitivity Streptococcus streptokinase, 47 kDa protein that dissolves blood clots Complexes with plasminogen to
44. Decreasing protein sensitivity Streptococcus streptokinase, plasmin sensitivity domain Attacks at Lys59 and Lys382, near each end
45. Protein Engineering - Applications Site-directed mutagenesis -> used to alter a single property Problem : changing
46. Protein Engineering – Applications Directed Evolution
47. Protein Engineering – Applications Directed Evolution
48. Protein Engineering – Applications Directed Evolution
49. Protein Engineering – Applications Directed Evolution
50. Protein Engineering – Directed Evolution
51. Protein Engineering - Applications
53. Скачать презентацию

Слайд 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

Слайд 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

Слайд 4

Mutagenesis Comparison of cellular and invitro mutagenesis

Слайд 5

Applications of directed mutagenesis

Слайд 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

Слайд 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

Слайд 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

Слайд 9

Protein Engineering Obtain a protein with improved or new properties

Слайд 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……..

Слайд 11

Site-directed mutagenesis methods
Old method
-> used before oligonucleotide –directed mutagenesis
Limitations:
->

just C-> T mutations
-> randomly mutated

Слайд 12

Site-directed mutagenesis methods

Слайд 13

Site-directed mutagenesis methods – Oligonucleotide - directed method

Слайд 14

Site-directed mutagenesis methods – PCR based

Слайд 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

Слайд 16

Слайд 17

General Directed Evolution Procedure
Random mutagenesis methods

Слайд 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 20129 (7x 10168) independent clones
Consequence -> not all modifications possible
-> modifications just along an evolutionary path !!!!

Слайд 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 !!!

Слайд 20

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 !!!

Typical Directed Evolution Experiment

Слайд 21

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

Слайд 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!!!

Слайд 23

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

Слайд 24

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

Слайд 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)

Слайд 26

Random Mutagenesis (PCR based) DNA Shuffling
DNase I treatment (Fragmentation, 10-50 bp,

Mn2+)

Reassembly (PCR without primers, Extension and Recombination)

PCR amplification

Слайд 27

Random Mutagenesis (PCR based) Family Shuffling
Genes coming from the same gene

family -> highly homologous
-> Family shuffling

Слайд 28

Random Mutagenesis (PCR based)

Слайд 29

Directed Evolution Difference between non-recombinative and recombinative methods
Non-recombinative methods
recombinative methods -> hybrids

(chimeric proteins)

Слайд 30

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 …..)

Слайд 31

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

Слайд 32

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

Слайд 33

Protein Engineering
Design of Thermal and Environmental stability:
Stabilization of α-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 α-Helix)
Introduction of disulfide bridges
Reduction of the unfolded state entropy with
X → Pro mutations

Слайд 34

Protein Engineering - Applications
Engineering Stability of Enzymes – T4 lysozyme
-> S-S

bonds introduction

Слайд 35

Protein Engineering - Applications
Engineering Stability of Enzymes – triosephosphate isomerase from

yeast

-> replace Asn (deaminated at high temperature)

Слайд 36

Protein Engineering - Applications
Engineering Activity of Enzymes – tyrosyl-tRNA synthetase from

B. stearothermophilus

-> replace Thr 51 (improve affinity for ATP) -> Design

Слайд 37

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

Слайд 38

DNA shuffling
JCohen. News note: How DNA shuffling works. Sci

293:237 (2001)
Maxygen, PCR without synthetic primers
Using family of related genes, digest into fragments
Heat and renature randomly
Use as PCR primers

Слайд 39

Altering multiple properties: rapid high-throughput screening
ex., subtilisin
Use 26 different

subtilisin genes
Shuffle DNA, construct library of 654 clones, and Tf B. subtilis
Assay in microtiter plates: originals plus clones
Activity at 23C; thermostability; solvent stability; pH dependence
Of 654 clones, 77 versions performed as well as or better than parents at 23C
Sequencing showed chimeras; one has 8 crossovers with 15 AAc substitutions

Слайд 40

Laundry, detergent and mushrooms
Peroxidase, ink cap mushroom; dye transfer inhibitor
Wash

conditions: bleach-containing detergents, pH 10.5, 50C,
high peroxide concentration (inactivates peroxidase)
Random mutagenesis or error-prone PCR, followed by DNA shuffling
One construct had 114x increase in thermal stability, 2.8x increase in oxidative stability

Слайд 41

ex., Coprinus cinereus heme peroxidase (ink cap mushroom); 343 AAc,

heme prosthetic group
Multiple rounds of directed evolution to generate mutant for dye transfer inhibitor in laundry detergent
Native form or WT is rapidly inactivated under laundry conditions at pH 10.5,
50C and high peroxide concentrations (5-10mM)
Combined mutants from site-directed and random mutagenesis led to mutant with
110x thermal stability, 2.8x oxidative stability
Additional in vivo shuffling of pt mutations -> 174x thermal stability and 100x oxidative stability
Cherry…Pedersen. 99. Nat Biotech “Directed evolution of a fungal peroxidase”

Mushroom peroxidase

Слайд 42

Molecular analysis of hybrid peroxidase

Слайд 43

Decreasing protein sensitivity
Streptococcus streptokinase, 47 kDa protein that dissolves blood

clots
Complexes with plasminogen to convert to plasmin, which degrades fibrin in clots
Plasmin also degrades streptokinase [feedback loop]
In practice, need to administer streptokinase as a 30-90 min infusion [heart attacks]
A long-lived streptokinase may be administered as a single injection

www-s.med.uiuc.edu; JMorrissey: Med Biochem 10/30/06

Слайд 44

Decreasing protein sensitivity
Streptococcus streptokinase, plasmin sensitivity domain
Attacks at Lys59 and

Lys382, near each end of protein
Resultant 328 AAc peptide has ~16% activity
Mutate Lys to Gln
Gln has similar size/shape to Lys also no charge
Single mutations similar to double to native in binding and activating plasminogen;
In plasmin presence, half-lives increased with double as 21x more resistant to cleavage
TBD…(2003) longer life wanted

Слайд 45

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

Слайд 46

Protein Engineering – Applications Directed Evolution

Слайд 47

Protein Engineering – Applications Directed Evolution

Слайд 48

Protein Engineering – Applications Directed Evolution

Слайд 49

Protein Engineering – Applications Directed Evolution

Слайд 50

Protein Engineering – Directed Evolution

Слайд 51

Directed Mutagenesis and Protein Engineering

Содержание

MutagenesisMutagenesis -> change in DNA sequence -> Point mutations or large

Consequences of point mutations within a coding sequence (gene) for the

Mutagenesis Comparison of cellular and invitro mutagenesis

Applications of directed mutagenesis

General strategy for directed mutagenesisRequirements: DNA of interest (gene or

Approaches for directed mutagenesis -> site-directed mutagenesis -> point mutations in

Protein Engineering-> Mutagenesis used for modifying proteinsReplacements on protein level ->

Protein Engineering Obtain a protein with improved or new properties

Rational Protein Design ⇒ Site –directed mutagenesis !!!Requirements: -> Knowledge of sequence

Site-directed mutagenesis methodsOld method -> used before oligonucleotide –directed mutagenesisLimitations: ->

Site-directed mutagenesis methods

Site-directed mutagenesis methods – Oligonucleotide - directed method

Site-directed mutagenesis methods – PCR based

Directed Evolution – Random mutagenesis-> based on the process of natural

General Directed Evolution ProcedureRandom mutagenesis methods

Directed Evolution LibraryEven a large library -> (108 independent clones) will

Limitation of Directed EvolutionEvolutionary path must exist - > to be

Successful experiments involve generally less than 6 steps (cycles)!!!Why?Sequences with improved

Evolutionary MethodsNon-recombinative methods: -> Oligonucleotide Directed Mutagenesis (saturation mutagenesis) -> Chemical

Evolutionary Methods Type of mutation – Fitness of mutantsType of mutations:Beneficial mutations

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-> PCR with low fidelity

Random Mutagenesis (PCR based) DNA ShufflingDNase I treatment (Fragmentation, 10-50 bp,

Random Mutagenesis (PCR based) Family ShufflingGenes coming from the same gene

Random Mutagenesis (PCR based)

Directed Evolution Difference between non-recombinative and recombinative methodsNon-recombinative methodsrecombinative methods -> hybrids

Protein EngineeringWhat can be engineered in Proteins ?-> Folding (+Structure):1. Thermodynamic

Protein EngineeringWhat can be engineered in Proteins ?-> Function:1. Binding (Interaction

Protein EngineeringFactors which contribute to stability:Hydrophobicity (hydrophobic core) Electrostatic Interactions:

Protein EngineeringDesign of Thermal and Environmental stability:Stabilization of α-Helix Macrodipoles Engineer

Protein Engineering - ApplicationsEngineering Stability of Enzymes – T4 lysozyme-> S-S

Protein Engineering - ApplicationsEngineering Stability of Enzymes – triosephosphate isomerase from

Protein Engineering - ApplicationsEngineering Activity of Enzymes – tyrosyl-tRNA synthetase from

Protein Engineering - ApplicationsEngineering Ca-independency of subtilisinSaturation mutagenesis -> 7 out

DNA shuffling JCohen. News note: How DNA shuffling works. Sci

Altering multiple properties: rapid high-throughput screening ex., subtilisinUse 26 different

Laundry, detergent and mushroomsPeroxidase, ink cap mushroom; dye transfer inhibitorWash

ex., Coprinus cinereus heme peroxidase (ink cap mushroom); 343 AAc,

Molecular analysis of hybrid peroxidase

Decreasing protein sensitivityStreptococcus streptokinase, 47 kDa protein that dissolves blood

Decreasing protein sensitivityStreptococcus streptokinase, plasmin sensitivity domainAttacks at Lys59 and

Protein Engineering - ApplicationsSite-directed mutagenesis -> used to alter a single

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

Похожие презентации

Mutagenesis
Mutagenesis -> change in DNA sequence
-> Point mutations or large

General strategy for directed mutagenesis
Requirements:
DNA of interest (gene or

Approaches for directed mutagenesis
-> site-directed mutagenesis
-> point mutations in

Protein Engineering
-> Mutagenesis used for modifying proteins
Replacements on protein level ->

Rational Protein Design
⇒ Site –directed mutagenesis !!!
Requirements:
-> Knowledge of sequence

Site-directed mutagenesis methods
Old method
-> used before oligonucleotide –directed mutagenesis
Limitations:
->

Directed Evolution – Random mutagenesis
-> based on the process of natural

General Directed Evolution Procedure
Random mutagenesis methods

Directed Evolution Library
Even a large library -> (108 independent clones)
will

Limitation of Directed Evolution
Evolutionary path must exist - > to be

Successful experiments involve generally
less than 6 steps (cycles)!!!
Why?
Sequences with improved

Evolutionary Methods
Non-recombinative methods:
-> Oligonucleotide Directed Mutagenesis (saturation mutagenesis)
-> Chemical

Evolutionary Methods Type of mutation – Fitness of mutants
Type of mutations:
Beneficial mutations

Random Mutagenesis (PCR based) Error –prone PCR
-> PCR with low fidelity

Random Mutagenesis (PCR based) DNA Shuffling
DNase I treatment (Fragmentation, 10-50 bp,

Random Mutagenesis (PCR based) Family Shuffling
Genes coming from the same gene

Directed Evolution Difference between non-recombinative and recombinative methods
Non-recombinative methods
recombinative methods -> hybrids

Protein Engineering
What can be engineered in Proteins ?
-> Folding (+Structure):
1. Thermodynamic

Protein Engineering
What can be engineered in Proteins ?
-> Function:
1. Binding (Interaction

Protein Engineering
Factors which contribute to stability:
Hydrophobicity (hydrophobic core)
Electrostatic Interactions:

Protein Engineering
Design of Thermal and Environmental stability:
Stabilization of α-Helix Macrodipoles
Engineer

Protein Engineering - Applications
Engineering Stability of Enzymes – T4 lysozyme
-> S-S

Protein Engineering - Applications
Engineering Stability of Enzymes – triosephosphate isomerase from

Protein Engineering - Applications
Engineering Activity of Enzymes – tyrosyl-tRNA synthetase from

Protein Engineering - Applications
Engineering Ca-independency of subtilisin
Saturation mutagenesis -> 7 out

DNA shuffling
JCohen. News note: How DNA shuffling works. Sci

Altering multiple properties: rapid high-throughput screening
ex., subtilisin
Use 26 different

Laundry, detergent and mushrooms
Peroxidase, ink cap mushroom; dye transfer inhibitor
Wash

Decreasing protein sensitivity
Streptococcus streptokinase, 47 kDa protein that dissolves blood

Decreasing protein sensitivity
Streptococcus streptokinase, plasmin sensitivity domain
Attacks at Lys59 and

Protein Engineering - Applications
Site-directed mutagenesis -> used to alter a single