Directed Evolution Methods for Protein Engineering · 2020-06-23 · Rational directed evolution...
Transcript of Directed Evolution Methods for Protein Engineering · 2020-06-23 · Rational directed evolution...
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Biosciences, Life Sciences Solutions,
Geneart AG, Regensburg, Germany
Directed Evolution Methods for Protein Engineering
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Directed Evolution
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Random mutagenesis vs. Rational design
Directed evolution by random mutagenesis The gene of interest is amplified using PCR conditions that enhance the natural error rate of the polymerase.
• Only a small subset of amino acid substitutions is accessible per site (6-8)
• Mutation rate cannot be varied per region within the gene
• No control over occuring substitutions
• Stop mutations will be generated
GeneArt® Directed Evolution technologies Synthetically constructed libraries can be rationally randomised. Only desired mutations appear, in predefined ratios.
• All possible substitutions are accessible
• Mutation rate can be precisely controlled in different regions
• Precise definition of included/excluded substitutions
• No technology related stop codon creation
Common challenges: Solution:
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GeneArt® technologies provide options to adapt protein properties to specific requirements
Affinity (e.g., affinity maturation of therapeutic antibodies)
Detergent resistance (e.g., create detergent-resistant laundry enzymes)
Enantioselectivity ( e.g., create enzymes that only catalyze the production of one stereoisomere)
Thermostability (e.g., make laundry enzymes work at 60°C )
Stability (e.g., raise the serum stability of protein drugs)
Specific activity (e.g., raise the activity of an industrial enzyme to help generate more product faster)
Others ...
Solubility (e.g., create soluble forms of rather insoluble proteins)
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How does directed evolution work?
Mutation Selection (screening)
Bottleneck (library generation):
Thought experiment:
A library of a protein with 300 aa and 20 random substitutions has a diversity of 7 x 1077.
(more possible mutants than atoms in the observable universe)
Bottleneck (screening):
• RNA display max 1012
• Phage display 109-1010
• Enzyme assays 103-104
→ Limit the diversity to a manageble but still meaningful size
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Rational directed evolution workflow
GeneArt® Site-Saturation Mutagenesis
• Systematically screen all single substitution variants of a target protein to identify beneficial substitutions
Diversity: 300 aa protein = 300 x 19 = 5,700
GeneArt® Combinatorial Libraries
• Combination of all beneficial substitutions to screen for synergies and an even better protein
e.g., 5 sites with 3 substitutions each = 35 = 243
Outcome
• Higher specific enzyme activity
• Improved enzyme thermostability
• Etc.
Screen
Screen
Improved variant
Wild type
Aim: Rationally limit the size of the library (diversity)
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Rational directed evolution workflow
Screen
Improved variant
Wild type
Shortcut:
• Knowledge gained from structural data
• Knowledge gained from homology data
• Literature
• Etc.
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Case study: Random vs. Rational approach
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Randomisation: whole open reading frame 23 selected amino acids No of mutations: average of 4 per construct average of 4 per construct Technology:
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....|....|....|....|....|....|....|....|
wildtype GVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICT
clone A01 GVVPILVELDGDVNGHKFSVFGEGEGDATYGKLTLKFICT
clone A02 GVVLILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFIGT
clone A03 GVVPILVELDGDLTGHKFSVSGEGEGDATYGKLTLKFICT
clone A04 GVVPILVELDGDVNGHKFSVSAEGECDATYGKLTLKFICT
clone A05 GIVPILVELDGDVNGHKFPVSGEGEGDATYGKLTLKFICT
clone A06 GVVPILVKLDGDVNAHKFSVSGQGEGDATYGKLTLKFICT
clone A07 GVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKAICT
clone A08 GVVPILVELEGDVNGHKFSVSGEGEGDASYGKLTLKFICT
.... ....
clone H12 GVVPILVELDGDVNGHKFSVSGEREGDATYGKLTLKFICT
Comparison of a random and a rational approach
Error-prone PCR Rationally designed library
50 60 70 80
....|....|....|....|....|....|....|....|
library GVVPILVELDGDVNGHXXSVSGEGEGDATXGKLTLXXICT
clone A01 GVVPILVELDGDVNGHRQSVSGEGEGDATGGKLTLRHICT
clone A02 GVVPILVELDGDVNGHAGSVSGEGEGDATEGKLTLLKICT
clone A03 GVVPILVELDGDVNGHIYSVSGEGEGDATLGKLTLIGICT
clone A04 GVVPILVELDGDVNGHLKSVSGEGEGDATPGKLTLQDICT
clone A05 GVVPILVELDGDVNGHMFSVSGEGEGDATWGKLTLLPICT
clone A06 GVVPILVELDGDVNGHSKSVSGEGEGDATSGKLTLCNICT
clone A07 GVVPILVELDGDVNGHRYSVSGEGEGDATVGKLTLDIICT
clone A08 GVVPILVELDGDVNGHGLSVSGEGEGDATKGKLTLPEICT
···· ····
clone H12 GVVPILVELDGDVNGHPMSVSGEGEGDATAGKLTLNAICT
Goal: Create new GFP phenotypes
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Design of the rational library
Lysine (K) abolishes dimerisation Chromophore
Shaded aa: remain wild type (wt); coloured aa: randomised
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Rational sesign vs. Error-prone PCR: Observed phenotypes
90.7 % 97 % green / regular 9.3 % non -
fluorescent
3 % green / bright
24 % green / faint
42 % green / regular
17 % green / bright
3 % blue / regular
14 % blue / bright
99.1 % 0.9 %
non -
fluorescent
90.7 % 97 % green / regular 9.3 % non -
fluorescent
3 % green / bright n = 3,482
90.7 % 97 % green / regular 9.3 % non -
fluorescent
3 % green / bright
24 % green / faint
42 % green / regular
17 % green / bright
3 % blue / regular
14 % blue / bright
99.1 % 0.9 %
non -
fluorescent
n = 5,859
24 % green / faint
42 % green / regular
17 % green / bright
3 % blue / regular
14 % blue / bright
99.1 % 0.9 %
non -
fluorescent
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Wild type
Blue
Rational design vs. Error-prone PCR
Sequence of the chromophore
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Rational design vs. Error-prone PCR: Summary
• The error-prone PCR library contains 10 times more functional variants due to neutral mutations. Just one “new” phenotype was identified.
• The rationally designed library contains more loss-of-function variants but 4 “new” phenotypes.
For you, this results in:
• Higher probability of creating the desired phenotype at all
• Less screening effort
• Less screening time
• Decreased cost
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Rational directed evolution workflow
Screen
Screen
Improved variant
Wild type
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GeneArt® site-saturation mutagenesis
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GeneArt® site-saturation mutagenesis
Replacement of the wild type (wt) residue by (up to) all 19 non-wild type amino acids
Different formats available: • All 19 non-wt variants per site as single clones • Average of 16 non-wt variants as single clones • Pool of all 19 non-wt variants per site • Pool of all single-substitution variants in one tube
All delivered as glycerol stocks (DNA preparations optional)
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RNA-Polymerase Subunit mjA´ of
Methanocaldococcus jannaschii:
Analysis of bridge helix region
Tan L. et al. Journal of Biology 2008 Imperial College London
Loss of function
Gain of function
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
GeneArt® site-saturation mutagenesis: Functional mapping of RNA polymerase (customer example)
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GeneArt® Combinatorial Libraries
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CTGATGGGGNNSATGAGGCCG
TCC
TCG
AGC
GCC
GCG
ATG
CAC
………
TAG Stop
GeneArt® Combinatorial Libraries
Ser
Ala
Met
His
Randomisation by NNS Randomisation using TRIM technology
CTGATGGGGxxxATGAGGCCG
TCC Ser
ATG Met
GCT Ala
CAC His
...
All amino acids appear
No customized randomization
Stop codons appear
Prone to out-of-frame mutations
Only desired amino acids present
Full control over amino acid distribution
No technology related stop codon introduction
Fewer of out-of-frame mutations
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500 503 506 509 512 515 518 521 524 527 530 533 536 539 542 545
A 0% 0% 0% 3% 0% 0% 8% 0% 21% 0% 5% 0% 7% 0% 100% 0%
C 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0%
D 0% 0% 0% 5% 0% 19% 7% 17% 0% 0% 6% 0% 7% 0% 0% 100%
E 0% 0% 0% 7% 0% 0% 6% 0% 0% 0% 8% 0% 4% 0% 0% 0%
F 0% 0% 0% 5% 0% 0% 0% 0% 22% 0% 7% 0% 4% 0% 0% 0%
G 0% 0% 0% 6% 0% 17% 8% 18% 21% 100% 7% 0% 7% 0% 0% 0%
H 0% 0% 0% 5% 0% 0% 7% 0% 19% 0% 0% 0% 7% 0% 0% 0%
I 0% 0% 0% 7% 100% 0% 0% 0% 0% 0% 9% 0% 3% 0% 0% 0%
K 0% 0% 0% 5% 0% 0% 6% 0% 0% 0% 9% 0% 4% 0% 0% 0%
L 0% 0% 0% 7% 0% 0% 7% 0% 0% 0% 6% 0% 7% 0% 0% 0%
M 0% 0% 0% 3% 0% 0% 8% 0% 0% 0% 5% 0% 5% 0% 0% 0%
N 0% 0% 0% 6% 0% 18% 6% 0% 0% 0% 9% 0% 7% 0% 0% 0%
P 0% 0% 0% 4% 0% 0% 8% 0% 0% 0% 0% 0% 7% 0% 0% 0%
Q 0% 0% 0% 3% 0% 0% 9% 0% 0% 0% 0% 0% 5% 0% 0% 0%
R 0% 0% 0% 5% 0% 18% 8% 18% 0% 0% 4% 0% 6% 0% 0% 0%
S 0% 0% 100% 5% 0% 18% 0% 14% 18% 0% 6% 0% 5% 0% 0% 0%
T 0% 0% 0% 6% 0% 0% 7% 0% 0% 0% 0% 100% 4% 0% 0% 0%
V 0% 100% 0% 3% 0% 16% 0% 17% 0% 0% 5% 0% 7% 0% 0% 0%
W 100% 0% 0% 6% 0% 0% 0% 18% 0% 0% 6% 0% 7% 0% 0% 0%
Y 0% 0% 0% 7% 0% 0% 7% 0% 0% 0% 6% 0% 3% 100% 0% 0%
# 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0%
GeneArt® Combinatorial Libraries: TRIM technology
• Possibility of complete customisation of permitted amino acids per position
• Even distribution or non-stoichiometric
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improved variant wild type
20-fold improvement of kcat/Km value
AtzA
Dechlorination
Scott et al. Appl Environ Microbiol 2009, 75:2184-2191. CSIRO Entomology
GeneArt® Combinatorial Libraries Catalytic improvement and evolution of Atrazine Chlorohydrolase
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Deliverables
Option 1 – amplified library 1. Non-amplified library, 17 to 170 fmol (1010 - 1011 specimen) 2. Amplification primers 3. >2 µg amplified library Option 2 – cloned library 1. Non-amplified library, 17 to 170 fmol 2. Amplification primers 3. >30 µg plasmid DNA 4. 12 aliquots of 0.5 mL glycerol stocks
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GeneArt® Library quality control
• Real-time quantitation of non-amplified library → up to 170 fmol (1011)
• Sequencing of amplified library → verify degenerated and constant regions
• Verification of transformation → up to 109 cfu
• Peer group sequencing of up to 96 clones → statistical peer group analysis
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How to order
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How to order GeneArt® directed evolution products
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How to order GeneArt® directed evolution products
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How to order GeneArt® directed evolution products
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Fill in questionnaire and email it to: [email protected]
How to order GeneArt® directed evolution products
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Acknowledgements
Synthetic
Biology
R&D Team
Carlsbad
Synthetic
Biology
R&D Team
Regensburg
Synthetic Biology
Software Team
Singapore
MIT - Collaboration
Dept Biological Engineering
Chris Voigt, Ron Weiss
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