Directed Evolution Methods for Protein Engineering

Transcription

1 Directed Evolution Methods for Protein Engineering XXX Biosciences, Life Sciences Solutions, Geneart AG, Regensburg, Germany The world leader in serving science 1

2 Directed Evolution 2

3 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. GeneArt Directed Evolution technologies Synthetically constructed libraries can be rationally randomised. Only desired mutations appear, in predefined ratios. Common challenges: 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 Solution: All possible substitutions are accessible Mutation rate can be precisely controlled in different regions Precise definition of included/ecluded substitutions No technology related stop codon creation 3

4 GeneArt technologies provide options to adapt protein properties to specific requirements Thermostability (e.g., make laundry enzymes work at 60 C ) Specific activity (e.g., raise the activity of an industrial enzyme to help generate more product faster) Affinity (e.g., affinity maturation of therapeutic antibodies) Stability (e.g., raise the serum stability of protein drugs) Solubility (e.g., create soluble forms of rather insoluble proteins) Enantioselectivity ( e.g., create enzymes that only catalyze the production of one stereoisomere) Others... Detergent resistance (e.g., create detergent-resistant laundry enzymes) 4

5 How does directed evolution work? Mutation Selection (screening) Bottleneck (library generation): Thought eperiment: A library of a protein with 300 aa and 20 random substitutions has a diversity of (more possible mutants than atoms in the observable universe) Bottleneck (screening): RNA display ma Phage display Enzyme assays Limit the diversity to a manageble but still meaningful size 5

6 Rational directed evolution workflow Aim: Rationally limit the size of the library (diversity) GeneArt Site-Saturation Mutagenesis Systematically screen all single substitution variants of a target protein to identify beneficial substitutions Diversity: 300 aa protein = = 5,700 Screen Screen Improved variant Wild type 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 = 3 5 = 243 Outcome Higher specific enzyme activity Improved enzyme thermostability Etc. 6

7 Rational directed evolution workflow Screen Shortcut: Knowledge gained from structural data Knowledge gained from homology data Literature Etc. Improved variant Wild type 7

8 Case study: Random vs. Rational approach The world leader in serving science 8

9 Comparison of a random and a rational approach Goal: Create new GFP phenotypes 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 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 Randomisation: whole open reading frame 23 selected amino acids No of mutations: average of 4 per construct average of 4 per construct Technology: Error-prone PCR Rationally designed library 9

10 Design of the rational library Shaded aa: remain wild type (wt); coloured aa: randomised Lysine (K) abolishes dimerisation Chromophore 10

11 Rational sesign vs. Error-prone PCR: Observed phenotypes 90.7 % 9.3 % 97 % green / regular non - fluorescent n = 3,482 3 % green / bright 99.1 % non - fluorescent 0.9 % n = 5, % green / faint 42 % green / regular 17 % green / bright 3 % blue / regular 14 % blue / bright 11

12 Rational design vs. Error-prone PCR Sequence of the chromophore Wild type Blue 12

13 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 13

14 Rational directed evolution workflow Screen Screen Improved variant Wild type 14

15 GeneArt site-saturation mutagenesis The world leader in serving science 15

16 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) 16

17 GeneArt site-saturation mutagenesis: Functional mapping of RNA polymerase (customer eample) RNA-Polymerase Subunit mja of Methanocaldococcus jannaschii: Analysis of bridge heli region Loss of function Gain of function Tan L. et al. Journal of Biology 2008 Imperial College London 17

18 GeneArt Combinatorial Libraries The world leader in serving science 18

19 GeneArt Combinatorial Libraries Randomisation by NNS CTGATGGGGNNSATGAGGCCG TCC TCG Ser AGC GCC Ala GCG ATG Met CAC His TAG Stop All amino acids appear No customized randomization Stop codons appear Prone to out-of-frame mutations Randomisation using TRIM technology CTGATGGGGATGAGGCCG TCC Ser ATG Met GCT Ala CAC His... Only desired amino acids present Full control over amino acid distribution No technology related stop codon introduction Fewer of out-of-frame mutations 19

20 GeneArt Combinatorial Libraries: TRIM technology 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% Possibility of complete customisation of permitted amino acids per position Even distribution or non-stoichiometric 20

21 GeneArt Combinatorial Libraries Catalytic improvement and evolution of Atrazine Chlorohydrolase Dechlorination AtzA improved variant 20-fold improvement of k cat /K m value wild type Scott et al. Appl Environ Microbiol 2009, 75: CSIRO Entomology 21

22 Deliverables Option 1 amplified library 1. Non-amplified library, 17 to 170 fmol ( 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 aliquots of 0.5 ml glycerol stocks 22

23 GeneArt Library quality control Real-time quantitation of non-amplified library up to 170 fmol (10 11 ) Sequencing of amplified library verify degenerated and constant regions Verification of transformation up to 10 9 cfu Peer group sequencing of up to 96 clones statistical peer group analysis 23

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29 Acknowledgements Synthetic Biology R&D Team Carlsbad Synthetic Biology R&D Team Regensburg MIT - Collaboration Dept Biological Engineering Chris Voigt, Ron Weiss Synthetic Biology Software Team Singapore 29

30 2014 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. 30

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