Directed evolution intense Flashcards
(50 cards)
Draw the iterative cycle of DE
See diagram
What are the conditions of EP-PCR
Increased MgCl2/Taq
Longer extension time
Altered nucleotide concentrations
Draw DNA shuffling
See diagram
How many codons can Trp be changed too?
5 other amino acids and 1 stop codon
Different methods for a phenotype/genotype link
In vivo:
Cell (Yeast/E. coli) or phage transfected with the DNA so can recover as plasmid or PCR
In vitro: No transfection so bigger but harder Ribosome display mRNA display Cis DNA approach Oil/water droplet approach
What are the two proteins you can fuse your protein onto in phage M13?
gp3 or gp8
How many copies of gp8 are there, size and what is it best for?
50aa
3000 copies
Peptides
How many copies of gp3 are there, size and what is it best for?
406aa
5 copies
Proteins
What is the max size for in vivo techniques?
10^10
What is the max size for in vitro techniques?
10^14
Draw ribosome display
See diagram
Draw mRNA display
See diagram
Example of selection
Chorimate mutase
Chorimate mutase selection example
Chorismate –> prephenate in Shikimate pathway for Tyr/Phe
Wanted to make a hexamer from WT dimer so introduced linkers however it was a worse enzyme
Set up a growth assay and did 2x rounds of random mutagenesis in CM deficient strain and generated a trimer with better activity (still worse than WT)
As it could grow they could not improve the enzyme as the system was not sensitive enough
Developed a system where the expression was reduced and the turnover rate was increased which produced an improved variant
Screening example - Fungal peroxidase
EP-PCR was used to mutate the WT gene to try and increase residual activity under ‘mild washing machine conditions’
In yeast perox is secreted and the residual activity was measured
64,000 variants where 2 were hits - contained common mutation of E239G which is not near the AS so would not have been found doing SDM
Screening example - Cytochrome P450 monooxygenase
Potential high selectivity in chemical transformations by oxygen incorportation in pharmaceuticals, chemical and detoxifications.
Issues with low turnover rates with unnatural substrates, low stabilty and they have to use electron-donating co-factors e.g. O2 and NADPH.
Aim to evolve efficent P450 with naphthalene which uses H2O2 instead.
The evolved a P450-camphor from P. Putida which had little activity with napthalene
Screened 32,000 by EP-PCR in a fluroscence based screen
3 variants had improvements and all contained a common mutation of E331K some distance from the AS
APEX 2 example
Heme peroxidases are powerful tools in biotechnology e.g. horse-radish peroxidase in western blots/ELISAs etc
Problems with HRP as hard to express and have to add externally
Engineered APEX as a monogenic peroxidase reporter derived from dimeric pea or soybean ascorbate peroxidases - lacks DS and Ca2+ binding sites so can be expressed in cells. Used for specific protein imaging in EM and proteomic mapping
Problem that APEX is not efficient so used DE
EP-PCR of APEX in yeast display system - adding the substrate biotin-phenol which if acted on labels the cell with biotin
More biotin = more activity and can sort using FACs
2nd and 3rd rounds they increased the selective pressure by targetting heme
Found 2x mutants - APEX2 and V/G-APEX which shared a common mutation. All of the mutations were found away from the active site
APEX 2 is used in EM/light cell microscopy using DAB as a substrate and proteomic tagging in live cells with MS analysis
Savinase - family shuffling example
Leading commercial Subtilisin found in washing powders
Segments of subtilisins were cloned by PCR from 25 natural Bacilli and cloned in the context of the savinase gene (63% identities). Cloned into the N/C terminals of savinase and shuffled
Hierarchical screening of milk then grew those with activity and assayed the supernatant with a Casein-derivative (FLURO)
Looked at if they could combine multiple properties temp, pH and solvent stability and they could
Best thermostable variant had 32aa so would not hvae been found due to the numbers in combination
Metagenomics search approach
4 soil samples isolated 94 substilisins with 32 mutations
52 could be expressed
Searched for (R) selective transaminases which gave 21 new sequences where 17 were true transaminases with (R) selectivity
Can use shuffling to improve further
Designing a Kemp eliminase
Well studied reaction - model system for the proton transfer from a carbon
Design was based on raising something to the TS
TIM was used as the scaffold and did 17 rounds of mutagenesis (EP-PCR and DNA shuffling) and selection allowed them to create a good enzyme which had high complementary to the TS
Alignment of the catalytic base was optimal so it could attack at the right position and the introduction of other groups allowed the stabilisation of the developing negative charge to push into generation
3DM - Esterase example
3DM is a commercial structure based sequence alignment and analysis tool
- identifies the distribution of aa within a protein super-family
Reduces the library size
Esterase - 4 residues were targetted - 240x fold increase in activity and E from 3 to 80
Designing a Diel-Alder
Reaction involves a series of different chemical bonds and stereocentres being formed at the same time
No naturally occurring enzyme
Rosetta methodology was used to design ideal AS based on the substrates and what they need
Used computer methods to design active sites and scan for scaffolds
207 stable scaffolds which allowed the backbone geometries - they selected a total of 84 for experimental validation where 50 were soluble in E. coli
Measured Diels-Alder by LC tandem-MS where 2 had activity
Further mutagenesis using crowd sourcing (Fold it) where amino acids interaction with the ligand were changed (10x) and remodelling of the loop regions into H-T-H motif (18x)
Split GFP
Example of protein only
Engineered a split GFP which did not affect protein solubilty of a normal protein
The tag is inaccessible on insoluble protein but accessible on soluble so can be used as a way of screening soluble variant proteins for further analysis
Adhirons properties (10)
Simple Single chain No DS bonds Extremely stable (tempa nd pH) Easy to engineer Can insert Cys for multimers High affinity Controlled specificity High yields Fast selection
