Protein engineering 2

Question 1

What is screening?

Answer 1

Assaying all mutants for desirable improved qualities

Question 2

What is selection?

Answer 2

Initially perform a step that removes the inactive proteins

before screening

Question 3

What are the two types of selection?

Answer 3

Biochemical selection

Selection for binding

Question 4

What is biochemical selection?

Answer 4

A cell will die and not form a colony without being transformed

Question 5

What is the sequence space problem?

Answer 5

It is not possible to store billions of DNA sequences

Question 6

What type of screening works well for hydrolytic enzymes?

Answer 6

Colorimetric or fluorescence detection

Can hydrolyse fluorescent substrates allowing them to fluoresce and changes to be detected

Question 7

What is an example of a Colorimetric or fluorescence detection for hydrolytic enzymes?

Answer 7

O-Nitrophenol (Absorbance: 400-420nm, turns yellow when the group is released)
Methyl Umbelliferone (Fluorescence: Ex 365 nm, Em 455 nm)

Question 8

How is pipetting of cells into wells sped up?

Answer 8

Robots

Question 9

What methods sorts 1000-2000 cells a second by levels of fluorescence?

Answer 9

Flow cytometry

Question 10

What changes which way a cell will fall in flow cytometry?

Answer 10

Electronic pules in response to levels of fluorescence

Question 11

In flow cytometry what do you do if the fluorescence is secreted?

Answer 11

coat the cell to encapsulate the fluorescence

Question 12

What is microfluidics?

Answer 12

Miniaturized lab processes (that involve fluids)

e.g. flow cytometry on a small scale

Question 13

In microfluidics does every oil droplet contain a cell?

Answer 13

No, some droplets will not contain cells.

Droplets are introduced more frequently than cells to prevent more than one cell being in a droplet

Question 14

What is the primary microfluidics?

Answer 14

Cheap and fast

Question 15

What is selection by display?

Answer 15

Display the library on something so that you there is a link between phenotype and genotype

Question 16

How does phage display work?

Answer 16

Fuse library with p3 gene (protein coat) so that the POI is expressed on the surface of the phage
These can then be used for binding assays

Question 17

What are the advantages of phage display?

Answer 17

fusion doesn’t affect infectivity so can increase population with E.coli, stable particles, high rate of phage synthesis

Question 18

What is the disadvantage of phage display?

Answer 18

Because the protein is propagated in E.coli it is biased towards proteins that are non-toxic to E.coli

Question 19

What is bacteria display?

Answer 19

Fuse protein to an outer membrane protein (e.g. ompA in E.coli)
These are then assayed for binding

Question 20

If your protein is small where might you insert it in bacterial display?

Answer 20

In a loop region of a surface protein

Question 21

If your protein is large where might you insert it in bacterial display?

Answer 21

Inserted terminally which may require a truncation to achieve

Question 22

Why might you choose bacterial display over phage display?

Answer 22

Phage display requires one extra step because you have to propagate library in E coli
Lower risk

Question 23

What is the primary advantage of yeast surface display?

Answer 23

Convenient for proteins that requires eukaryotic PTM’s for folding

Question 24

How does yeast display work?

Answer 24

Normally fuse Aga2p to the protein
Aga1p is naturally on the surface of the protein
Aga2p is secreted and binds to Aga1 via disulphide bonds
A reducing agent can therefore be used to elute the Aga2-POI

Question 25

How might you visualise whether the POI is expressed on the surface and whether it is able to interact with the binding partner?

Answer 25

Tag the binding partner (maybe with fluorescently tagged avidin)
Tag the POI (maybe with GFP)

In this example high green and low red would show lots of POI expressed but binding partners not able to bind

Question 26

What is ribosome display?

Answer 26

Library with no stop codon is subjected to transcription and translation
The protein never disengages with the ribosome
Hence protein, mRNA and ribosome is all captured together
Selection, elution, reverse transcription, repeat

Question 27

What are the advantages of ribosome display?

Answer 27

Not limited by transformation efficiency, no toxicity bias, ~1 day per cycle

Question 28

What are the disadvantages of ribosome display?

Answer 28

No PTM, mRNA not very stable – lots of RNases on skin