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Презентация на тему Directed Mutagenesis and Protein Engineering

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MutagenesisMutagenesis -> change in DNA sequence -> Point mutations or large modificationsPoint mutations (directed mutagenesis):Substitution: change of one nucleotide (i.e. A-> C)Insertion: gaining one additional nucleotideDeletion: loss of one nucleotide
Directed Mutagenesis  and  Protein Engineering MutagenesisMutagenesis -> change in DNA sequence -> Point mutations or large modificationsPoint Consequences of point mutations within a coding sequence (gene) for the proteinSilent Mutagenesis  Comparison of cellular and invitro mutagenesis Applications of directed mutagenesis General strategy for directed mutagenesisRequirements: DNA of interest (gene or promoter) must Approaches for directed mutagenesis -> site-directed mutagenesis Protein Engineering-> Mutagenesis used for modifying proteinsReplacements on protein level -> mutations 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: -> just 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 evolution- General Directed Evolution ProcedureRandom mutagenesis methods Directed Evolution LibraryEven a large library -> (108 independent clones) will not Limitation of Directed EvolutionEvolutionary path must exist - > to be successful Successful experiments involve generally less than 6 steps (cycles)!!!	Why?Sequences with improved properties Evolutionary MethodsNon-recombinative methods:   -> Oligonucleotide Directed Mutagenesis (saturation mutagenesis) 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 (chimeric proteins) Protein EngineeringWhat can be engineered in Proteins ?-> Folding (+Structure):	1. Thermodynamic Stability Protein EngineeringWhat can be engineered in Proteins ?-> Function:	1. Binding (Interaction of Protein EngineeringFactors which contribute to stability:	 Hydrophobicity (hydrophobic core) Electrostatic Interactions: Protein EngineeringDesign of Thermal and Environmental stability:	 Stabilization of -Helix Macrodipoles Engineer Protein Engineering - ApplicationsEngineering Stability of Enzymes – T4 lysozyme-> S-S bonds introduction Protein Engineering - ApplicationsEngineering Stability of Enzymes – triosephosphate isomerase from yeast-> Protein Engineering - ApplicationsEngineering Activity of Enzymes – tyrosyl-tRNA synthetase from B. Protein Engineering - ApplicationsEngineering Ca-independency of subtilisinSaturation mutagenesis -> 7 out of DNA shuffling JCohen. News note: How DNA shuffling works. Sci 293:237 Altering multiple properties: rapid high-throughput screening ex., subtilisinUse 26 different subtilisin Laundry, detergent and mushroomsPeroxidase, ink cap mushroom; dye transfer inhibitorWash conditions: ex., Coprinus cinereus heme peroxidase (ink cap mushroom); 343 AAc, heme Molecular analysis of hybrid peroxidase Decreasing protein sensitivityStreptococcus streptokinase, 47 kDa protein that dissolves blood clotsComplexes Decreasing protein sensitivityStreptococcus streptokinase, plasmin sensitivity domainAttacks at Lys59 and Lys382, Protein Engineering - ApplicationsSite-directed mutagenesis -> used to alter a single propertyProblem 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
Слайды презентации

Слайд 2 Mutagenesis
Mutagenesis -> change in DNA sequence

-> Point

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

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

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

(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


Слайд 4 Mutagenesis Comparison of cellular and invitro mutagenesis

Mutagenesis Comparison of cellular and invitro mutagenesis

Слайд 5 Applications of directed mutagenesis

Applications of directed mutagenesis

Слайд 6 General strategy for directed mutagenesis
Requirements:
DNA of

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

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

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

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

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

Protein Engineering  Obtain a protein with improved or new properties

or new properties


Слайд 10 Rational Protein Design
 Site –directed mutagenesis !!!


Requirements:

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



-> 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

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

–directed mutagenesis

Limitations:
-> just C-> T mutations
-> randomly mutated



Слайд 12 Site-directed mutagenesis methods

Site-directed mutagenesis methods

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

Site-directed mutagenesis methods – Oligonucleotide - directed method

Слайд 14 Site-directed mutagenesis methods – PCR based

Site-directed mutagenesis methods – PCR based

Слайд 15 Directed Evolution – Random mutagenesis
-> based on the

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

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 17 General Directed Evolution Procedure
Random mutagenesis methods

General Directed Evolution ProcedureRandom mutagenesis methods

Слайд 18 Directed Evolution Library
Even a large library -> (108

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

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 -

Limitation of Directed EvolutionEvolutionary path must exist - > to be

> 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

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

(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

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

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

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

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

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

mutagenesis)


Слайд 24 Random Mutagenesis (PCR based) with degenerated primers (saturation

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

mutagenesis)


Слайд 25 Random Mutagenesis (PCR based) Error –prone PCR
-> PCR

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

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

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

(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

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

the same gene family -> highly homologous

-> Family shuffling


Слайд 28 Random Mutagenesis (PCR based)

Random Mutagenesis (PCR based)

Слайд 29 Directed Evolution Difference between non-recombinative and recombinative methods
Non-recombinative methods
recombinative

Directed Evolution Difference between non-recombinative and recombinative methodsNon-recombinative methodsrecombinative methods -> hybrids (chimeric proteins)

methods -> hybrids (chimeric proteins)


Слайд 30 Protein Engineering
What can be engineered in Proteins ?

->

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

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 ?


->

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

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

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

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

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

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


Слайд 34 Protein Engineering - Applications
Engineering Stability of Enzymes –

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

T4 lysozyme
-> S-S bonds introduction


Слайд 35 Protein Engineering - Applications
Engineering Stability of Enzymes –

Protein Engineering - ApplicationsEngineering Stability of Enzymes – triosephosphate isomerase from

triosephosphate isomerase from yeast
-> replace Asn (deaminated at high

temperature)

Слайд 36 Protein Engineering - Applications
Engineering Activity of Enzymes –

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

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

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

-> 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

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

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.,

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

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;

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

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

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

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

Molecular analysis of hybrid peroxidase

Слайд 43
Decreasing protein sensitivity
Streptococcus streptokinase, 47 kDa protein

Decreasing protein sensitivityStreptococcus streptokinase, 47 kDa protein that dissolves blood

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

Decreasing protein sensitivityStreptococcus streptokinase, plasmin sensitivity domainAttacks at Lys59 and

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

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

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

Protein Engineering – Applications Directed Evolution

Слайд 47 Protein Engineering – Applications Directed Evolution

Protein Engineering – Applications Directed Evolution

Слайд 48 Protein Engineering – Applications Directed Evolution

Protein Engineering – Applications Directed Evolution

Слайд 49 Protein Engineering – Applications Directed Evolution

Protein Engineering – Applications Directed Evolution

Слайд 50 Protein Engineering – Directed Evolution

Protein Engineering – Directed Evolution

Слайд 51 Protein Engineering - Applications

Protein Engineering - Applications

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