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Jonathan Sun
University of Illinois at Urbana Champaign BIOE 506
February 15, 2010
http://www.sliceofscifi.com/wp-content/uploads/2008/02/nc_evolution_080103_ms.jpg
Outline
Introduction Motivation Methods Applications Conclusions
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Evolution
Darwin => natural selection 1970 – John Maynard Smith
Evolution is a walk from one functional protein to another in the landscape of all possible sequences
“Fitness” of protein based on favorability for reproduction or based on experimenter in artificial selection
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Romero and Arnold: Exploring Protein Fitness Landscapes by Directed Evolution
Picture (not many more to come)
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Screening criteria is important
Stability can be used instead of improvement
Allows for functionally neutral mutations
Romero and Arnold: Exploring Protein Fitness Landscapes by Directed Evolution
What is Directed Evolution? An engineering strategy used to improve
protein functionality through repeated rounds of mutation and selection
First used in the ‘70s Around .01-1% of all random mutations
estimated to be beneficial Based off natural evolution processes,
but in a much quicker timescale
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Another (more direct?) Method Rational design – modify protein
function based on understanding consequences of certain changes
We are still relatively ignorant as to how a protein’s gene sequence encodes functionality
Directed evolution avoids this problem by creating libraries of variants possessing desired properties
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Why is it Needed?
Biotechnology – increased demand for specific properties that don’t necessarily occur naturally
Can be used to improve existing proteins’ functionality
Can be applied as far as the ideas come – enzymes and catalysts to pharmaceuticals or crops
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Successful Directed Evolution Desired function should be/have:
Physically feasibleBiologically or evolutionarily feasibleLibraries of mutants complex enough to
contain rare beneficial mutationsRapid screen to find desired function
Increases understanding of protein function and evolution – disconnects protein from natural context
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Basic Method
A parent gene is selected Mutations/diversity are induced
(mutagenesis or recombination) Selection criteria applied Repeat with new parent genes selected
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Bloom and Arnold: In the light of directed evolution: Pathways of adaptive protein evolution
Random Mutagenesis
Traditional method Point mutation based – error prone PCR Frequency of beneficial mutations very
low Multiple mutations virtually impossible to
come out positive
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DNA Shuffling
Recombination used to create chimeric sequences containing multiple beneficial mutations
“Family shuffling” of homologous genes “Synthetic shuffling” – oligonucleotides
combined to create full-length genes Whole-genome shuffling – accelerated
phenotypic improvements Drawback – high homology required
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RACHITT
Random Chimeragenisis on Transient Templates
Small DNA fragments hybridized on a scaffold to create a chimeric DNA fragment
Incorporates low-homology segments
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Even More Methods Assembly of Designed Oligonucleotides
(ADO) Mutagenic and Unidirectional Reassembly
(MURA) Exon Shuffling Y-Ligation-Based Block Shuffling Nonhomologous Recombination – ITCHY,
SCRATCHY, SHIPREC, NRR Combining rational design with directed
evolution
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ADO
Nonconserved regions with conserved parts as linkers
PCR with dsDNA without primers Full length genes in expression vector Creates large diversity of active variants
without codon bias for parental genes
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MURA
Random fragmentation of parental gene Reassembled with unidirectional primers
for specific restriction site Generates N-terminally truncated DNA
shuffled libraries
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Exon Shuffling
Similar to natural splicing of exons Chimeric oligos mixed together,
controlling combination of which exons to be spliced
Protein pharmaceuticals based on natural human genes – less immune response
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Nonhomologous Recombination Creation of new protein folds Structures not present in nature – useful for
evolution of multifunctional proteins Incremental truncation for the creation of
hybrid enzyme (ITCHY) – two genes in expression vector with unique restriction sites, blunt end digestion, ligated ->SCRATCHY
Nonhomologous random recombination – potentially higher flexibility in fragment size and crossover frequency
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A Combination
Rational design with directed evolution Success depends on ability to predict
fitness of a sequence Computationally demanding Kuhlman et al created a new protein fold Focuses library diversity for directed
evolution
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Directed Evolution in Action Has been applied to improve
polymerases, nucleases, transposases, integrases, recombinases
Applications in genetic engineering, functional genomics, and gene therapy
Optimized fluorescent proteins and small-molecule probes for imaging and techniques like FRET
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The Case of a Fluorescent Protein dsRED – parent protein evolved to have
better solubility and shorter maturation time
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dsRed mCherry
Biochemical Catalysts
Useful in industry because of high selectivity and minimal energy requirements
Need for high availability at low costs Active and stable under process
conditions – not naturally occuring Some reaction enzymes still yet to be
identified and produced
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Application to Enzymes Improve stability and activity of biochemical
catalysts Can modify pH or temperature dependence Substrate specificity or catalytic activity MANY applications:
Proteolytic – Subtilisin in detergentsCellulolytic and esterases – biofuel productionCytochrome P450 superfamily – catalyze
hydroxilation Whole metabolic pathway evolution
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Whole Metabolic Pathways Closer to natural compound production Single enzyme activity upregulation
does not necessarily lead to increase in final product
Different methods:Whole genome shufflingKey enzymes targetedNaturally expressed operons targetedTarget gene regulation factors
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Pharmaceuticals
Therapeutic proteins Antibodies – natural somatic
recombination Vaccines – improved effectiveness, less
side effects Viruses – gene therapy and vaccine
development
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Agriculture Plants with increased tolerance for
herbicides or expression of toxins Golden rice
Expresses elevated beta-carotene (Vitamin A precursor)
Directed evolution - 23 times more insecond version
Not approved for distribution
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http://en.wikipedia.org/wiki/Golden_rice
Conclusions Directed evolution can be a powerful
tool taking advantage of nature’s power to improve upon itself
Used in a wide variety of applications for protein improvement – stability, activity, substrate specificity, etc
Potential for genetically engineering improved drugs or crops
Ultimately, combining tools will lead to better understanding and applications
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Thank You!
Questions?
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