McPherson Lectures 1-3

Question 1

what is directed evolution

Answer 1

the creation of new protein activities through the principles of natural selection to chang: pH and thermal stability, optimum pH and temp, binding properties (i.e. km for substrates) and catalytic properties

Question 2

although the ultimate goal is to be able o design a protein de novo which performs a certain function, why do most directed evolution experiments explore a sample of proteins with activity

Answer 2

most random sequences won’t fold into a functional protein so existing proteins already have stable folds, display functional properties and may have minor activities which can be enhanced by sequence changes

Question 3

what are the methods for introducing variability

Answer 3

error prone per, DNA shuffling, family shuffling or random mutagenesis

Question 4

why is sparse sampling used in directed evolution experiments

Answer 4

possible sequence space = 1.3x10^55kg of protein = 2x mass of earth so sparse sampling just uses enough diversity to select interesting proteins

Question 5

how does error prone PCR work

Answer 5

reduce the PCR fidelity e.g. using non-proof reading Taq polymerase, high conc of mgcl2, increased extension time or altered relative nucleotide conc to introduce variability

Question 6

how many mutation per codon are produced in error prone pcr

Answer 6

1

Question 7

why is recombination usually performed after error prone pcr

Answer 7

it is unlikely to lead to concerted accumulation of beneficial mutations

Question 8

describe the process of DNA shuffling

Answer 8

1. fragment DNA by physical method e.g. dnase 1
2. heat denaturing allows random annealing which self prime upon addition of DNA polymerase. repeat cycle to build up size of whole gene
3. resulting genes can be amplified by PCR to form a variant library which can be cloned into a vector
4. screen for desired activity then carry out saturation mutagenesis to improve

Question 9

why can error prone pcr/dna shuffling not be used to explore complete sequence space

Answer 9

limited by codon usage- with 1 mutation/codon tryptophan, for e.g., can only be mutated to 5 aa

Question 10

how are protein coding sequences isolated once the desired protein is selected for

Answer 10

a physical association between the expressed protein and its coding protein has to be created. in vivo it can then be transfected into a cell then cells are grown and DNA coding sequence is recovered as plasmid DNA or by PCR. in vitro can be done via ribosome display and reverse transcription, mRNA display or micro fluids encapsulation

Question 11

what are 3 possible methods for selection

Answer 11

survival phenotypes, fluorescence selection, chromogenic enzyme activities

Question 12

how can we overcome the problem of not being able to select for “better” mutants if you find something that allows the cell to grow

Answer 12

downregulate protein expression or introduce a degradation signal to select for mutants which allow growth under these pressures

Question 13

describe screening

Answer 13

pick individual colonies, put them in a 96 well plate, grow, lyse and assay for activity

Question 14

what is the problem with screening

Answer 14

low throughput, inefficient and expensive if most variants are inactive

Question 15

which substrate does Cyt p450 usually use

Answer 15

camphor

Question 16

which substrate did they try to change cyt p450 to use

Answer 16

naphthalene

Question 17

describe the first stage of the directed evolution experiments performed on cytochrome p450

Answer 17

error prone PCR was used to generate 200,000 clones of which 32000 were screened using fluorescence and found 3 variants

Question 18

which 3 variants were further tested following error prone pcr and fluorescence screening of cyt p450

Answer 18

M7-6H which had 11 fold higher activity and contained the mutations R280L and E331K, M7-4H 5 fold higher activity and E331K mutation and M7-8H with 5 fold higher activity and mutations E331K and C242F

Question 19

where was the E331K mutation discovered by directed evolution situated

Answer 19

not near the active site- would not have been an obvious choice

Question 20

what is APEX

Answer 20

a haem peroxidase

Question 21

why was directed evolution carried out on APEX

Answer 21

it can be used for intracellular specific protein imaging by EM and spatially resolved proteomic mapping but it isn’t as efficient as one might like

Question 22

describe the experiment to produce APEX2 via directed evolution

Answer 22

first a base enzyme was generated which can be expressed in cytosol. they then generated a library of variants using error prone pcr, cloned them into ecoli then transformed into yeast. treated with substrate to label the variants then used FACS to select for improved activity. in 2nd and 3rd round increased selection pressure was applied to test for efficient haem incorporation. found APEX2 and VPGAPEX and in both most mutations were away from the active site

Question 23

what was the major mutation in APEX2

Answer 23

A134P

Question 24

what are the applications of APEX2

Answer 24

as reporter to generate contrast for EM/VM, mass spec analysis

Question 25

describe the process of family shuffling

Answer 25

it mimics natural evolution, recombining reated sequences from different organisms to create new “functional variant” sequence combinations

Question 26

what is the benefit of family shuffling

Answer 26

recombining to create new variants should creat functional proteins so few colonies can be screened and the vast majority will be functional. in addition family shuffling can maintain one quality whilst enhancing another

Question 27

describe the experiment used to create Savinase, the leading commercial subtilisin

Answer 27

segments of subtilisins were cloned by PCR from 25 natural bacilli. they retained the N and C coding ends but swapped the majority of the coding region in between using shuffling between aa 60-224. these variants were then screened using a skimmed milk screening assay and found most were inactive but selected for the most active. repeated but with a casein screening assay. casein is quenched upon cleavage by a protease. this screen produced 654 clones which were then screened for activity at 23c, thermostability, solvent stability and pH dependence. the mutant which was most thermostable was suaveness which contained 32 mutations compared to most thermostable parent.

Question 28

what are the 3 possible rational approaches

Answer 28

saturation mutagenesis, designed enzyme and computational structure based design

Question 29

describe the trinuclrotide approach in saturation mutagenesis

Answer 29

you only add 1 codon per amino acid so you get a small library with relatively equal numbers of each amino acid

Question 30

what is the CAST approach

Answer 30

combinatorial active site saturation test

Question 31

how does CASTing work

Answer 31

you look for combinations of mutations in the active site which give an improvement

Question 32

describe how the enzyme design approach was used to create HG3, a kemp eliminase

Answer 32

began with xylanase scaffold and computationally designed 11 point mutations, the most successful of which was HG13.17 which was created after 17 rounds of mutagenesis and screening. it has high shape complementarity between the TS and active site and 3 groups which stabilise the developing negative charge in TS

Question 33

what is the basis for computational structure based design

Answer 33

computationally identify and predict residues that are important in catalysis

Question 34

describe the use of computational structure based design to generate a diels-alder enzyme

Answer 34

it decided on a catalytic mechanism and ideal active site including a binding pocket for the 2 substrates in the correct relative orientation with suitable hydrogen bond donors and acceptors. rosetta methodology was used to design in silicon enzyme models with active sites with the desired properties, searching 207 stable protein scaffold for backbone geometrics which allow the 2 catalytic residues and substrates. a total of 84 designs were selected for experimental validation then genes for these were synthesised with a his tag and expressed in e.coli. 50 were soluble and purified and Diels-alder activity was measured by LC-tandem MS. 2 designs were found to have activity then crown sourcing and further mutagenesis experiments was used to generate improved enzymes