Professional Documents
Culture Documents
Bio 5 Tec Protein Engineering
Bio 5 Tec Protein Engineering
Bio 5 Tec Protein Engineering
CC BY 4.0
PROTEIN
ENGINEERING
MANIPULATING PROTEIN
STRUCTURE AND FUNCTION FOR
BIOTECHNOLOGY
- DR THOMAS
CC BY 4.0
SHAFEE
OUTLINE
WHAT PROTEIN ENGINEERING IS
‐ Background and introduction
Definition
Aims
Applications
COMBINED APPROACHES
‐ The best of both worlds
2
WHAT PROTEIN
ENGINEERING
IS
DEFINITION | AIMS | APPLICATIONS
- DR THOMAS
CC BY 4.0
SHAFEE
PROTEIN ENGINEERING
noun: protein engineering
Techniques … to manipulate the structure and function of a
protein so that it acquires specific desired properties
noun: genetic engineering
The alteration of the genome of an organism by laboratory
techniques
AIMS OF PROTEIN
ENGINEERING
Catalysis
Temperature Specificity
Activity
pH Sterioselectivity
Environ- Regulation
Salinity
Solvent
ment
Sequence
Temperature
Host organism
Express Solvent
Solubility
pH
Characterise stability
Salt
Proteolysis
Substrate Solve structure
Regulation Function
Binding partners Allergenicity
Toxicity
Immunogenicity
APPLICATIONS OF PROTEIN
ENGINEERING
PHARMACEUT
AGRICULTURE RESEARCH
ICALS
WHITE
BIOENERGY
BIOTECH
Images: Wikimedia commons 6
RATIONAL
DESIGN
METHODS | EXAMPLES | LIMITATIONS
- DR THOMAS
CC BY 4.0
SHAFEE
THE RATIONAL
DESIGN
PROCESS
Knowledge
‐ Based on protein
knowledge
Structure
Mechanism Hypothesis
Dynamics
Natural variation
‐ Analogous to
mechanical Modification
engineering
8
- DR THOMAS
CC BY 4.0
SHAFEE
TOPICS
Active site
modification
Split
Disulphides
proteins
Chemical
modification
Fusion
proteins Cyclisation
Bio- Computer
informatics modelling
9
- DR THOMAS
CC BY 4.0
SHAFEE
CHEMICAL MODIFICATION
‐ Formaldahyde Formaldehyde
Extensive modification ⟶ Inactivated toxoid production
‐ PEGylation
Flexible hydrophilic coat ⟶ Solubility
Poly-ethylene glycol
Reduced accessibility ⟶ Protease resistance and non-antigenicity
Increased size ⟶ Serum half-life
‐ Fluorophores
Fluorescein
Fluorescent labelling ⟶ Tracking location or dynamics
Selenocysteine
‐ Considerations
Exposure of modified residues
Original function of modified residues
10
- DR THOMAS
CC BY 4.0
SHAFEE
AIM
Reduce immunogenicity
IMMUNOGENICTY Increase serum half-life
Uricase 10kDa
‐ Attached PEG polymers Uric acid Poly-ethylene glycol
ENGINEERI
Lysine coupling
+
Allantoin
‐ Optimised PEG number and length
Maximise improvements
NG
‐
Also improved solubility at neutral pH
Also increased serum half-life
Mutation
SITE-DIRECTED Primer
DN
MUTAGENESIS A
‐ Modified PCR
Whole plasmid Linear increase Linear increase
Overlap extension
‐ Introduce point
mutations
‐ Introduce short Non-methylated DpnI - digested
insertions or
deletions
12
- DR THOMAS
CC BY 4.0
SHAFEE
FUSION PROTEINS
Gene 1
‐ Creation Gene 2
Remove stop codon of first gene
Ligate genes together in frame
Include linker codons
Gene 1 Linker Gene 2
‐ Aims
Combine the properties of the components
E.g. Addition of antibody Fc fragment to proteins increases their serum halflife
Co-localise the components
E.g. Set of enzymes that work in a reaction pathway
‐ Considerations
Linker length and flexibility
Ability for proteins rotate relative to each other
Distance between protein components
Protease resilience
Ability for domains to fold
13
- DR THOMAS
CC BY 4.0
SHAFEE
AIM
Increase processivity
PROCESSIVITY Retain fidelity and stability
‐ Fusion dsDNA
Polymerase
Pyrococcus furiosus DNA polymerase (Pfu) binder
ENGINEERING
‐ Generality
Also works with other polymerases
Template length (kb)
SPLIT PROTEINS
‐ Creation Original protein
‐ Considerations
Half-proteins must: fold independently
not spontaneously ‘dimerise’ Active split
be inactive when apart protein
bind and be active when brought together
15
- DR THOMAS
CC BY 4.0
SHAFEE
DISULPHIDES
‐ Creation
Mutation of two codons to cysteine
Protein kept in oxidising environment
‐ Aims
Stability enhancement
Enthalpy increase ≈ 3.5 kcal/mol
Entropy decrease ≈ Logarithm of trapped loop length
‐ Considerations
Inter-cysteine distance
Inter-cysteine orientation
Trapped loop length and flexibility
Original function of mutated residues
Original function of flexibility
Folding pathway of protein (multistep)
16
- DR THOMAS
CC BY 4.0
SHAFEE
CYCLISATION
Cyclisable
‐ Creation
‐ Aims
Thermostability
Up to 1.7 kcal/mol
Protease resistance N- to C- termini distance
Especially exopeptidase (Å)
AIM
Pain killer activity by specific binding to ion channels
‐ cMII-5
No longer folded or functional
Engineering stable peptide toxins by means of backbone cyclization: stabilization of the alpha-conotoxin MII, (R Clark 2005) 18
- DR THOMAS
CC BY 4.0
SHAFEE
19
- DR THOMAS
CC BY 4.0
SHAFEE
BIOINFORMATIC
APPROACHES
‐ Codon optimisation
Different organisms have different tRNA ratios
Matching codon frequency to host increases expression
Naturally
‐ Considerations existing
Altered codons can affect mRNA (stability, 2° structure, IRES) sequences
Increased translation rates can cause misfolding
Hypothesis
‐ Consensus sequence
Most mutations are mildly destabilising
Through genetic drift, homologues accumulate different mutations
Therefore consensus should be more stable than existing sequences Modification
‐ Considerations
Availability of homologous sequences
20
- DR THOMAS
CC BY 4.0
SHAFEE
AIM
‐
STABILITY Improving phosphorous bioavailability in animal feed
sequences
Starting
ENGINEERING
‐ Final TM = 78 °C
Crystal structure resolves loops too flexible to be seen in
natural phytases
OUTCOME
COMPUTATIONAL
MODELLING
‐ Considerations
Requires deep knowledge of reaction mechanism Modification
Requires extreme computational power
Simulation either ignores: quantum mechanism of active site
or structure and dynamics in rest of protein
22
- DR THOMAS
CC BY 4.0
SHAFEE
DE NOVO
ENZYME
DESIGN
‐ Disembodied
amino acids placed
to stabilise reaction
transition state
‐ Existing protein
structures searched
for backbones with
correct orientations
‐ Other residues in
active site
optimised for ‐ Theozymes
Theoretical enzyme
packing
Quantum mechanical modelling
AIM
‐
RETRO-ALDOLASE Retro-aldol reaction not performed by any known enzyme
O OH O O
‐ Theozyme Enzyme +
O
Amino acids positioned to increase reactivity of
O
25
INTERMISSION
DIRECTED
EVOLUTION
METHODS | EXAMPLES | LIMITATIONS
- DR THOMAS
CC BY 4.0
SHAFEE
THE
EVOLUTION
CYCLE
‐ Mimic natural
evolution
Single gene evolved in
cycle Screening (fitness
Mutagenesis differences)
(variation)
Gene amplification (heredity)
‐ Experimental
control over
Mutation rate
Environment
Selection pressure
‐ Protein
understanding not
required
TOPICS
Screening (fitness
Mutagenesis differences)
(variation)
Gene amplification (heredity)
Ensuring heredity
29
- DR THOMAS
CC BY 4.0
SHAFEE
UNDERSTANDING
MUTATIONS a
100%
b
100%
80% 80%
100%
60% 60%
noun: distribution of fitness effects 80%
Frequency
Frequency
40% 60%
40%
deleterious:neutral:beneficial mutations 0% 0%
0%
3
noun: epistasis
Trait value
2
30
- DR THOMAS
CC BY 4.0
SHAFEE
GENERATING
VARIATION
Starting gene
Point
mutations
‐ Point mutations
Error prone PCR
Random mutations
Whole gene or region
Insertions and
deletions
‐ Insertion/deletions
Difficult protocols
Large effects
‐ Shuffling
Requires similar genes Shuffling
Can use multiple genes
AIM
‐
CYTOCHROME P450 Used for oxidisation in white biotech
64 °C
retains oxidase activity
CONSIDERATIONS
‐ Mutation rate
Proportion of beneficial mutations
Likelihood of finding synergistic interactions
Avoiding multiple mutants all being deleterious
Expected screening/selection throughput
‐ Library size
Throughput of the screening/selection
Number of possible single or double mutations for the gene
Full coverage of library can guarantee that best available variant was found
‐ Types of mutations
Single nt mutations can’t convert a codon to all others
Shuffling introduces large numbers of mutations likely to be functional
33
- DR THOMAS
CC BY 4.0
SHAFEE
DETECTING FITNESS
DIFFERENCES
SELECTION SCREENING
Survival to next Survival to next
Desired activity Desired activity
round round
‐ Direct coupling
Quantitative Sorting above
assay threshold
‐ Indirect coupling
34
- DR THOMAS
CC BY 4.0
SHAFEE
SELECTION SYSTEMS
SELECTION
Survival to next
Desired activity
round
‐ Direct coupling
Immobilised target
A B C
‐ Binding
Immobilised substrate
Bind, wash, elute
‐ Cell survival
Toxic A B C
Genome kept the same except compound
target gene
Antibiotic resistance / Auxotrophy
Non-toxic
compound
35
- DR THOMAS
CC BY 4.0
SHAFEE
AIM
‐
TRANSPORTER Affects overall rate of bio-production of n-octane
‐ Surprise
promiscuous activity
47% improved n-octane efflux
400% improved α-pinene efflux
α-pinene
‐ Only one of the mutations in the channel of transporter
Directed evolution of an E. coli inner membrane transporter for improved efflux of biofuel molecules, (J L Foo 2013) 36
- DR THOMAS
CC BY 4.0
SHAFEE
SCREENING SYSTEMS
R OH
+ H2O
Enzym
+ R–H
SCREENING
e
‐ Microfluidics
Flow cytometry
Microdroplet emulsion
Images: Wikimedia commons 37
- DR THOMAS
CC BY 4.0
SHAFEE
CONSIDERATIONS
SELECTION SCREENING
High throughput High control
High sensitivity Information-rich
Usually cheap
‐ Microtitre plate
‐ Binding Low throughput
Versatile High sensitivity
Doesn’t require surrogate substrate Simple to create
Can’t assay catalysis
‐ Microfluidics
‐ Cell survival High throughput
Multiple rounds fast Low sensitivity
Simple to use but difficult to create Difficult to create
Difficult to tune dynamic range
38
- DR THOMAS
CC BY 4.0
SHAFEE
CONSIDERATIONS
BOTH SELECTION AND SCREENING
‐ You get what you screen for
Surrogate substrates may be different from true targets
Evolved variants mostly display the ‘easiest’ solution (most evolvable)
39
- DR THOMAS
CC BY 4.0
SHAFEE
ENSURING HEREDITY
COMPARTMENT COVALENT LINK
Gene Protein
Gene
Protein
40
- DR THOMAS
CC BY 4.0
SHAFEE
COMPARTM
ENT Compartment = Cell
Each cell expresses one gene variant
Substrate must be able to enter cell
Or protein displayed on cell surface
Each cell assayed (e.g. FACS)
Gene
Compartment = Microtitre plate
‐ Compartment Cells grown in clonal populations in plate wells
Protein and gene are co- Each population expresses one gene variant
localised either in vivo Protein Cells lysed to release protein
or in vitro Substrate added
Storage copy made
Cell lysate assayed
‐ In vivo examples
Cellular assays
Compartment = Microdroplet
‐ In vitro examples Each compartment contains
Gene variant
Microtitre plate
Transcription-translation (cellular or in vitro)
Micro-droplet sorting
Substrate
Each microdroplet assayed
41
- DR THOMAS
CC BY 4.0
SHAFEE
42
- DR THOMAS
CC BY 4.0
SHAFEE
AIM
Target = signalling or receptor protein
MATURATION Affinity (kd) < 1 nanomolar
Phage M13
‐ Phage display Phage
library
‐ Library size up to 1010 Gene library
Fusion of gene to viral coat protein gene Immobilised
target
ENGINEERING
CONSIDERATIONS
COMPARTMENTALISATION COVALENT LINKAGE
Substrate must enter compartment Can engineer toxic activities
Typically binding only
‐ In vivo
High protein expression ‐ mRNA/ribosome display
RNA not very stable
‐ In vitro
Can engineer toxic activities ‐ DNA display
High control over environment High control over environment
44
- DR THOMAS
CC BY 4.0
SHAFEE
EVOLUTION ROUNDS
‐ Selection consistency
Increase stringency
Alter selected properties
‐ Number of rounds
Improve upon last round
Find epistatic interactions
Diminishing returns (frequency and magnitude)
45
- DR THOMAS
CC BY 4.0
SHAFEE
RATIONALISING RESULTS
‐ Methods
Output
Measuring effects of mutations individually
Finding the minimal set of mutations that give the improvement
Removes ‘hitchhiking’ mutations
Structural and mechanistic studies
46
- DR THOMAS
CC BY 4.0
SHAFEE
BENEFITS LIMITATIONS
‐ Simple concepts ‐ Starting activity range
Requires some starting activity
‐ Widely applicable Can rarely improve native activity
‐ High success rate ‐ Requires high-throughput assay
Typically enzyme-specific
‐ Requires no knowledge of protein
Screens can be expensive
(or of mutations)
Synergistic mutations hard to find
47
- DR THOMAS
CC BY 4.0
SHAFEE
COMBINED
APPROACHES
CUTTING EDGE IDEAS
- DR THOMAS
CC BY 4.0
SHAFEE
BLENDED TECHNIQUES
SIMULTANEOUS
‐ Smart libraries
Maximising library fitness
Finding synergistic mutations
SEQUENTIAL
‐ Improving designed enzymes
Optimising crude designs
Compensating for structural disruption
49
- DR THOMAS
CC BY 4.0
SHAFEE
SMART LIBRARIES
‐ The problem with mutagenic libraries
High throughput screening is difficult and expensive
Beneficial mutations are rare
Prior knowledge Focussed mutagenesis
Synergistic mutations are even rarer
natural previous
DNA synthesis
variation experiment
‐ Optimise DFE proportions structure mechanism semi-random codons
Reduce mutations likely to be deleterious
Increase mutations likely to beneficial
Make multiple simultaneous mutations to find synergy
50
- DR THOMAS
CC BY 4.0
SHAFEE
AIM
For use as stereospecific esterase in white biotech
‐ Sterioselectivity
OUTCOME
IMPROVING DESIGNED
ENZYMES
Initial protein designs or modifications done rationally
‐ Compensatory mutations
Offset structural destabilisation
52
- DR THOMAS
CC BY 4.0
SHAFEE
AIM
‐
RETRO-ALDOLASE Retro-aldol reaction not performed by any known enzyme
O OH O O
‐ ‘Theozyme’ Enzyme +
O
Amino acids positioned to increase reactivity of
O
AIM
‐
RETRO-ALDOLASE Designed enzyme rate well below natural enzymes
SUMMARY
WHAT PROTEIN ENGINEERING IS
Manipulation of protein structure and function to acquire specific properties
COMBINED APPROACHES
Semi-rational ‘smart’ libraries (Simultaneous)
Improving upon previous designs (Sequential)
55
- DR THOMAS
CC BY 4.0
SHAFEE
[END]
‐ Recommended reading
Recent advances in engineering proteins for biocatalysis, (Y Li 2014)
Exploring protein fitness landscapes by directed evolution, (P Romero 2009)
Beyond directed evolution—semi-rational protein engineering and design, (S Lutz 2010)
56
- DR THOMAS
CC BY 4.0
SHAFEE
OPEN QUESTIONS TO
CONSIDER
57