Protein engineering 2 Flashcards Preview

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Flashcards in Protein engineering 2 Deck (28)
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1
Q

What is screening?

A

Assaying all mutants for desirable improved qualities

2
Q

What is selection?

A

Initially perform a step that removes the inactive proteins

before screening

3
Q

What are the two types of selection?

A

Biochemical selection

Selection for binding

4
Q

What is biochemical selection?

A

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

5
Q

What is the sequence space problem?

A

It is not possible to store billions of DNA sequences

6
Q

What type of screening works well for hydrolytic enzymes?

A

Colorimetric or fluorescence detection

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

7
Q

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

A
O-Nitrophenol (Absorbance: 400-420nm, turns yellow when the group is released)
Methyl Umbelliferone (Fluorescence: Ex 365 nm, Em 455 nm)
8
Q

How is pipetting of cells into wells sped up?

A

Robots

9
Q

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

A

Flow cytometry

10
Q

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

A

Electronic pules in response to levels of fluorescence

11
Q

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

A

coat the cell to encapsulate the fluorescence

12
Q

What is microfluidics?

A

Miniaturized lab processes (that involve fluids)

e.g. flow cytometry on a small scale

13
Q

In microfluidics does every oil droplet contain a cell?

A

No, some droplets will not contain cells.

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

14
Q

What is the primary microfluidics?

A

Cheap and fast

15
Q

What is selection by display?

A

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

16
Q

How does phage display work?

A

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

17
Q

What are the advantages of phage display?

A

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

18
Q

What is the disadvantage of phage display?

A

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

19
Q

What is bacteria display?

A

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

20
Q

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

A

In a loop region of a surface protein

21
Q

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

A

Inserted terminally which may require a truncation to achieve

22
Q

Why might you choose bacterial display over phage display?

A

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

23
Q

What is the primary advantage of yeast surface display?

A

Convenient for proteins that requires eukaryotic PTM’s for folding

24
Q

How does yeast display work?

A

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

25
Q

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

A

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

26
Q

What is ribosome display?

A

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

27
Q

What are the advantages of ribosome display?

A

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

28
Q

What are the disadvantages of ribosome display?

A

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