Directed Evolution Methods for Protein Engineering · 2020-06-23 · Rational directed evolution...

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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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Screen

Improved variant

Wild type

Shortcut:

• Knowledge gained from structural data

• Knowledge gained from homology data

• Literature

• Etc.


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:

50 60 70 80

....|....|....|....|....|....|....|....|

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


fluorescent

3 % green / bright n = 3,482


fluorescent

3 % green / bright

24 % green / faint


17 % green / bright

3 % blue / regular

14 % blue / bright

99.1 % 0.9 %

non -

fluorescent

n = 5,859

24 % green / faint


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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Screen

Screen

Improved variant

Wild type


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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CTGATGGGGNNSATGAGGCCG

TCC

TCG

AGC

GCC

GCG

ATG

CAC

………

TAG Stop


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


How to order

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How to order GeneArt® directed evolution products

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Fill in questionnaire and email it to: [email protected]


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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

Directed Evolution Methods for Protein Engineering · 2020-06-23 · Rational directed evolution...

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