SCALED BIOSYSTEMS
Research & Development of molecular systems for control of plant growth and morphogenesis
mazura@sci.muni.cz
Screening and characterization of novel β-glucosidases
for target probing of plant hormone system
Bioinformatics, E.coli transformation and screening,
DNA isolation + (basic molecular biology techniques)
Racionální design a řízená
evoluce proteinů
the Bigpicture
Protein structure
Protein Engineering
the Bigpicture
Protein Engineering
the Bigpicture
Biosystems
10E-10 → 10E7m
Scale is important !
Protein Engineering
Rational Design
(Directed Mutagenesis)
Random Mutagenesis/
Directed Evolution
Improved Protein
Protein Engineering
Google: AdvGentech5.ppt
Rational Design
(Directed Mutagenesis)
Bioinformatics
Lliterature search: current knowledge in this area,
data mining, identification of possible problems,
look for collaborations material etc.
DNA and AA sequence information:
composition, similarity,
physicochemical properties,
motifs, experimental design for directed mutagenesis
or directed evolution procedure
3D structure derived information:
structure motifs, similarity,
surfaces, canals and cavities,
check and evaluation of aminoacids
possible involved in misfolding
HotSpot Wizard 1.7
Google: AdvGentech5.ppt
Protein Engineering
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 …..)
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?
1. 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
Protein Engineering
Factors which contribute to stability:
1. Hydrophobicity (hydrophobic core)
1. Electrostatic Interactions:
-> Salt Bridges
-> Hydrogen Bonds
-> Dipole Interactions
1. Disulfide Bridges
1. Metal Binding (Metal chelating site)
1. Reduction of the unfolded state entropy with
X → Pro mutations
Protein Engineering
Design of Thermal and Environmental stability:
1. Stabilization of α-Helix Macrodipoles
1. Engineer Structural Motifes (like Helix N-Caps)
1. Introduction of salt bridges
1. Introduction of residues with higher intrinsic properties for their
conformational state (e.g. Ala replacement within a α-Helix)
1. Introduction of disulfide bridges
1. Reduction of the unfolded state entropy with
X → Pro mutations
Protein
Engineering
Rational Design
(Directed Mutagenesis)
Random Mutagenesis/
Directed Evolution
Improved Protein
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
- Deletion: loss of one nucleotide
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
Codon Usage is different in different species
Mutagenesis
Comparison of cellular and invitro mutagenesis
General strategy for
directed mutagenesis
Requirements:
- DNA of interest (gene or promoter) must be
cloned
- Expression system must be available -> for
testing phenotypic change
Applications of directed mutagenesis
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
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
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……..
Site-directed mutagenesis methods
Site-directed mutagenesis methods –
Oligonucleotide - directed method
Site-directed mutagenesis methods – PCR based
Protein
Engineering
Rational Design
(Directed Mutagenesis)
Random Mutagenesis/
Directed Evolution
Improved Protein
Protein Engineering
The concept of laboratory-directed protein evolution is not new.
Systematic approaches to directed evolution of proteins
have been documented since the 1970s
One early example is the evolution of the EbgA protein from Escherichia
coli, an enzyme having almost no -galactosidase activity.
Through intensive selection of a LacZ deletion strain of
E. coli for growth on lactose as a sole carbon source, the
wild-type EbgA was “evolved” as a -galactosidase sufficient to
replace the lacZ gene function (Campbell, J.1973).
Directed Evolution – Random mutagenesis
-> based on the process of natural evolution
- NO structural information required
- NO understanding of the mechanism required
General Procedure:
1. Generation of genetic diversity
⇒ Random mutagenesis
2. Identification of successful variants
⇒ Screening and seletion
Protein Engineering
Directed Evolution
Successful directed evolution has four requirements:
(i) the desired function should be physically feasible,
(ii) the function should be biologically or evolutionary feasible, i.e., a
mutational pathway must exist to get from an original protein to
tailored protein through ever-improving variants,
(iii) it should be possible to make libraries of mutants complex enough
to contain rare beneficial mutations and
(iv) a rapid screen or selection reflecting the desired function should
be available (Arnold,1998)
Approaches for directed random mutagenesis
-> 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
General Directed Evolution Procedure
Random mutagenesis methods
Limitation of Directed Evolution
1. Evolutionary path must exist - > to be successful
1. Screening method must be available
-> You get (exactly) what you ask for!!!
-> need to be done in -> High throughput !!!
Successful experiments involve generally
less than 6 steps (cycles)!!!
Why?
1. 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
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
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!!!
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 !!!
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)
Random Mutagenesis (PCR based)
DNA Shuffling
DNase I treatment (Fragmentation,
10-50 bp, Mn2+)
Reassembly (PCR without primers,
Extension and Recombination)
PCR amplification
Random Mutagenesis (PCR based)
Family Shuffling
Genes coming from the same
gene family -> highly
homologous
-> Family shuffling
Random Mutagenesis (PCR based)
Staggered Extension Process (StEP)
Sequence Homology-Independent Protein Recombination (SHIPREC)
Truncation for the Creation of Hybrid EnzYmes (ITCHY)
Directed Evolution
Difference between non-recombinative and recombinative methods
Non-recombinative methods
recombinative methods ->
hybrids (chimeric proteins)
...engineering proteins by
circular permutation
Protein engineering must not necessarily involve the
substitution of amino acids. The reorganization of a
proteins’ primary sequence can also change the catalytic
properties. We are using a technique called circular
permutation to explore the effects of termini relocation on
catalysis, as well as protein stability & dynamics.
...engineering proteins by circular permutation
lipase B from Candida antarctica, an
important biocatalyst in asymmetric
synthesis. Upon relocating the protein
termini by circular permutation, we
observe up to 175-fold enhanced
catalytic performance while preserving
the enzyme’s enantio-selectivity.
Stefan Lutz group
HTS (High throughput screening) SystemHTS (High throughput screening) System
Screening methodsScreening methods
• Genetic selection
• Growth
• Survival
• Display technology
• In-vitro display (cell-free translation)
• Phage display
• Cell surface display (Bacteria & Yeast)
• Solid or liquid-phase assay
Screening systemsScreening systems
• FACS ( Fluorescence-activated cell sorter)
• Digital image spectroscopy
• Fluorescence detection technique
Screening in E.coli
Theoretical implications for mutant library
construction (size of the library)
Standard Modified
Library size containing 95% of variants 12 53
Library size with 95% chance of being complete 17.4 77
Mutant library parameters calculated for our sample via standard equations
and modified by introducing empirical coefficient.
Number of transformants (L)=20 and
number of possible sequence variants (V)=4.
Slonomax
HTS (High throughput screening) SystemHTS (High throughput screening) System
Screening methodsScreening methods
• Genetic selection
• Growth
• Survival
• Display technology
• In-vitro display (cell-free translation)
• Phage display
• Cell surface display (Bacteria & Yeast)
• Solid or liquid-phase assay
Screening systemsScreening systems
• FACS ( Fluorescence-activated cell sorter)
• Digital image spectroscopy
• Fluorescence detection technique
H
T
S
H
T
S
H
T
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Microfluidics for HTS
Microfluidics for HTS
Todd Thorsen lab
High Content Analysis and High Content Screening
High Content Analysis (HCA) and High Content Screening (HCS) are imaging based multi-parametric
approaches of cell analysis at the single-cell level. Originally developed as a complementary technology to
traditional biochemical high-throughput screening (HTS) in drug discovery, today High Content Screening is
established in a far broader area of the life science space as an unbiased method of imaging multiple cellular
samples.
Miami Project High Content Screening Core
The core technology in the HCS uses the Cellomics VTI ArrayScan with a
Thermo Fisher robot to feed the scanner.
This system can scan about thirty 96 well plates over night, acquiring 9 fields per well,
with 3-4 colors. This is a lot of data.
Functional Diversity
parental enzymes
Activity at high pH
Activity at low pH
Activity in organic solvent
Thermostability
Increased catalytic efficiency
Stereoselectivity
New substrate specificity
Shuffle
Functional expression
Protein Engineering - Applications
Engineering Stability of Enzymes – T4 lysozyme
-> S-S bonds introduction
Protein Engineering - Applications
Engineering Stability of Enzymes – triosephosphate isomerase from yeast
-> replace Asn (deaminated at high temperature)
Protein Engineering - Applications
Engineering Activity of Enzymes – tyrosyl-tRNA synthetase from B.
stearothermophilus
-> replace Thr 51 (improve affinity for ATP) -> Design
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
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
Protein Engineering – Applications
Directed Evolution
Protein Engineering – Applications
Directed Evolution
Protein Engineering – Applications
Directed Evolution
GFP