Directed evolution intense

Question 1

Draw the iterative cycle of DE

Answer 1

See diagram

Question 2

What are the conditions of EP-PCR

Answer 2

Increased MgCl2/Taq
Longer extension time
Altered nucleotide concentrations

Question 3

Draw DNA shuffling

Answer 3

See diagram

Question 4

How many codons can Trp be changed too?

Answer 4

5 other amino acids and 1 stop codon

Question 5

Different methods for a phenotype/genotype link

Answer 5

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

Question 6

What are the two proteins you can fuse your protein onto in phage M13?

Answer 6

gp3 or gp8

Question 7

How many copies of gp8 are there, size and what is it best for?

Answer 7

50aa
3000 copies
Peptides

Question 8

How many copies of gp3 are there, size and what is it best for?

Answer 8

406aa
5 copies
Proteins

Question 9

What is the max size for in vivo techniques?

Answer 9

10^10

Question 10

What is the max size for in vitro techniques?

Answer 10

10^14

Question 11

Draw ribosome display

Answer 11

See diagram

Question 12

Draw mRNA display

Answer 12

See diagram

Question 13

Example of selection

Answer 13

Chorimate mutase

Question 14

Chorimate mutase selection example

Answer 14

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

Question 15

Screening example - Fungal peroxidase

Answer 15

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

Question 16

Screening example - Cytochrome P450 monooxygenase

Answer 16

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

Question 17

APEX 2 example

Answer 17

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

Question 18

Savinase - family shuffling example

Answer 18

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

Question 19

Metagenomics search approach

Answer 19

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

Question 20

Designing a Kemp eliminase

Answer 20

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

Question 21

3DM - Esterase example

Answer 21

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

Question 22

Designing a Diel-Alder

Answer 22

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)

Question 23

Split GFP

Answer 23

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

Question 24

Adhirons properties (10)

Answer 24

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

Question 25

Draw Adhiron

Answer 25

See diagram

Question 26

What are Adhirons based on?

Answer 26

Cystatins (cysteine protease inhbitors)
Consensus approach (most common aa's)

Question 27

Range of applications for Adhirons (6)

Answer 27

Affinity purifications without tags
Co-crystallisation
Inhibitors/Activators
Diagnostics
Western blots
Drug targeting

Question 28

How many aa can be inserted in the loops of Adhirons and what approach did they use?

Answer 28

18aa
Trinucelotide approach so get good representation of all aa (not cystine)
Loops better as constrained

Question 29

Phage and adhiron

Answer 29

M13 phage with adhiron cloned on truncated p3
Amber stop codon to allow expression as fusion or seperate
Easy phenotype and genotype link

Question 30

Draw affimer biopanning process

Answer 30

See diagram

Question 31

Adhiron - FcyRs example

Answer 31

Target in rheumatoid arthritis and inflammation
3 class of receptors where 3a and b only have 2aa differences
Biopanning was used to select binders against 3a which prevents IgG binding when it was present
XRC showed one binds competitively and one binds allosterically
Used XRC data to design 2 small molecules which bind to the receptor

Question 32

Adhiron - hSUMO example

Answer 32

No reagent to distigush between hSUMO 1 and 2
Recently AB for hSUMO1
Found binders aginst 1, 2 or both where ITC showed they were selective
hSUMO2 is involved in poly-Ub and when incubated with Adhiron it showed a reduction of PML bodies in vivo

Question 33

Adhiron - Grb2 example

Answer 33

SH2 domains are found in many proteins in the human proteome - involved in many aspects of cell signaling and role in cancer progression
Wanted to specifically target Grb2 which contains SH2 domains
ELISA to check specificity
Sequence showed typical SH2 consensus sequence but was highly specific to one SH2 domain
BEttwe than siRNA and chemicals which normally knock-down members of the same family

Question 34

Adhiron - small molecule TNT example

Answer 34

TNBsA is a version of TNT which reacts with primary amines so can be conjugated to proteins such as IgG and ovalbumin
Selected binders aginst TNT bound to ovalbumin and IgG which shows it is binding to something common
Went on to screen a range of TNT analogs which showed binding to some but not others showing it can discriminate at a molecular level

Question 35

Adhirons - biosensors

Answer 35

IL-8 biosensor was made and showed better sensitivity in less time. Potential to make multiple devices

Clostridum difficule is a global health issue where one risk is the misuse of antibiotics. 3 markers of the disease Toxin A, B and GDH
Adhirons were selected against them where SPR showed toxin A binder is specific with no binding to toxin B and they were sensitive with high affinity

Question 36

Examples of stereoisomers having different effects

Answer 36

Mint and carraway
Wilson’s disease (D)-penicilliamine for treating the disease and (L) is toxic
Thalidomide disaster

Question 37

Draw the reaction lipase catalyses

Answer 37

See diagram

Question 38

Lipase example

Answer 38

Wanted to make an enzyme which was either (R) or (S) specific
Did 5 rounds of EP-PCR looking at increases in E value which had 1 mutant per round
6th round they increased the mutation rate to see if they could further increase the E value and it had an extra 4 mutations
Draw lipase graph
History of how you get to generation 5 is important as it may not be the best route through seq space
Went back and did saturation mutagenesis and found the 1st generation was optimal however the second was not instead Phe was better
They then looked at the 3rd generation knowing that Phe was better which showed a higher E value
This showed saturating each position would have given better results later
They used the saturation mutant of generation 3 to do EP-PCR on which gave an E value of ~25 which is a significant increase of the 1.1 starting value
Always choosing higher E values

Question 39

Draw lipase example graph

Answer 39

See diagram

Question 40

What is backcrossing?

Answer 40

Can get rid of non-advantageous mutations by backcrossing with the parent gene

Question 41

Hydantonase example

Answer 41

Stereospecific for the isomer you don’t want
(D) isomer produces an acidic produce so can see the change using pH indicator
(L) no reaction
DE of 10,000 clones with (D) or (L) looking at activity where they successfully changed the enzyme from (D) to (L) - the single aa mutation I96F was enough
Also found better (D) specific enzymes in the 1st and 2nd rounds

Question 42

Tagatose 1,6 bisphosphate

Answer 42

DHAP + G3P = FBP or TBP
2 planes react together differently (Si and Si for FBP)
Started with TBP aldolase and carried out two round of DE. The first round used DNA shuffling and small increases in FBP were seen
Round 2 used DNA shuffling with the 3 round 1 mutants and assayed with 4x less substrate to increase stringency - only 1/800 showed improvements but TBP activity was similar to WT
Round 3 used DNA shuffling of the gen 2 variant where 1/3600 showed improved FBP but still preferred TBP
Looking at the Km/Kcat showed if you saturate the enzyme it always runs at Vmax so Km is irrelevant so if you saturate then you will always make FBP
Tested making FBP using 31-P NMR

Question 43

Principle of microscopic diversity

Answer 43

Same mechanism either way

Question 44

N-acetylneuraminic lyase

Answer 44

Converted pyruvate and N-acetylmannosamine into N-acetylneuraminic acid
Saturation mutagenesis at residue 192 showed the E192N mutant changes the specificity for a different aldehyde substrate showing slight selectivity (0.58) towards 4(S). The dipropyl amides are related to some antiflu products e.g. Tamiflu
EP-PCR of E192N and screened with (R) and (S) product in the cleavage directed showed that residues 167/208 control the stereoselectivity
(S) is slightly favored in terms of energetics but can favor the (R) with kinetics
QM-MM modeling showed WT does not distinguish between the two it can make both but it is T167 which is involved in the orientation of the molecules

Question 45

Lipase - CASTing

Answer 45

Reece’s group did EP-PCR to find hot spots and then saturation mutagenesis - needed 50,000 clones to find the hit E= 51
Then did computer modelling and found another 2 mutations involved in switching the stereochemistry E=63

CASTing was used look at combinations of saturation
Grouped the 6 aa around the Me group into 3 groups A/B/C.

First found used NN(G/T) = 3000 transformants (95%) where only B gave a hit E=8
The second round they used B as a template and (A/G/T)NT (reduced amino acids no leucines) Only got a hit from A but E=594 (10,000 clones)
Synergistic mutations

Question 46

Draw CASTing graph thing

Answer 46

See diagram

Question 47

David bake and computer algorithms

Answer 47

To model the TS and find existing proteins which can accommodate the active site
Has been successfully done however the activity is low therefore DE is used to increase
An artificial type 1 retro-aldolase was designed to cleave an artificial butanone substrate which is not found in biological systems. The active site was put onto 5 different scaffolds of the TIM family. Of the 72 designed tested in the lab 44% of these showed promising activity. Random mutagenesis was used to improve the enzyme 4,400x
DE caused the addition of a lysine into the substrate binding pocket and the next round removed the first one i.e. corrected the position

Question 48

Fructose 1, 6 bisphosphate aldolase - thermostability

Answer 48

DNA shuffling was used to improve the thermostability
240x higher half-life at 53oC than either parent
Enhanced activity in various polar and non-polar organic solvents
Example were you get more than you screen for

Question 49

NS1 monobody

Answer 49

NS1 monobody based on fibronectin type 3 domain scaffold
NS1 targets RAS - found dysregulated in 30% of tumors
Specifically, targets H and K-RAS but not N-RAS
Targets both the GTP and GDP-bound forms
Disrupts dimerisation in the alpha4, beta6, alpha 5 region

Question 50

Draw NMR experiment

Answer 50

See diagram