TB2-2: Where do you get your protein from? Flashcards

1
Q

Name 3 proteins that are abundant and highly enriched in their natural sources.

A
  • hemglobin (major protien component in blood)
  • lysozyme (an enzyme found in chicken egg white)
  • acetylcholine receptors (found in the electric organs (nervous system) of rays (fish))
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

Name an application where it is possible to obtain enough material from native sources? Name an example of a structure solved by this method.

A

cryo-electron microscopy (EM)
solved flagellar motor structure

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

Why can’t most proteins be obtained from their native source?

A

they aren’t sufficiently abundant in their natural source

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

If proteins aren’t sufficiently abundant in their natural sources, how do we get enough?

A

produce the proteins ourselves using heterologous expression systems

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

Other than producing enough of a portein for analysis, what other advantage does using an heterologous expression system have?

A

We can design the protein for how we want it be

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

Define “heterologous expression system”.

A

a type of cell culture system that can be easily transfected with a foreign gene

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

What are the 5 most commonly used expression systems?

A

E.coli
Yeast
Insect cells/Baculovirus
Mammalian cells

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

What are 4 advantages of using E.coli as an expression system?

A
  • easy to grow
  • inexpensive media
  • can produce large quantities quickly (few hours)
  • huge quantities if use fermenter chamber
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

What is the most commonly used promoter to drive expression of our protein?

A

T7 (phage) promoter

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

How does the T7 expression system work?

A

-T7 pol is present in the E.coli genome
- Expression of T7 is represses by the lac repressor
- (allolactose gets rid of repression; IPTG is an analogue of allolactose)
- IPTG removes lac repressor repression
- causing expression of T7 pol
- polymerase initiates expression of protein of interest

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

What strain of E. coli is commonly used for plasmid transformation?

A

BL21 strain

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

How are E.coli cells that have taken up the plasmid selected for?

A

Selection using antibodies

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

What are E.coli cells (as expression systems) grown in, in order to make lots of them?

A

Shaker culture or in a fermenter

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

When is IPTG generally added to E.coli expression systems? (i.e. at what point in during the growth phase of colonies)
How do we detect this phase?

A

At high quantities
- the optical density is reached when growing in the log/exponential growth phase of the bacterial growth curve

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

Why would we not introduce IPTG to E.coli expression system to induce T7 promoter expression before reaching the exponential growth phase?

A

Addition of IPTG diverts the cellular energy away from growth of the cell/replication, and instead towards making the protein of interest
- therefore more efficient to wait and have a large amount of cells making the protein of interest

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

up to fancy ecpression strains

A