TB2-2: Where do you get your protein from? Flashcards
Name 3 proteins that are abundant and highly enriched in their natural sources.
- 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))
Name an application where it is possible to obtain enough material from native sources? Name an example of a structure solved by this method.
cryo-electron microscopy (EM)
solved flagellar motor structure
Why can’t most proteins be obtained from their native source?
they aren’t sufficiently abundant in their natural source
If proteins aren’t sufficiently abundant in their natural sources, how do we get enough?
produce the proteins ourselves using heterologous expression systems
Other than producing enough of a portein for analysis, what other advantage does using an heterologous expression system have?
We can design the protein for how we want it be
Define “heterologous expression system”.
a type of cell culture system that can be easily transfected with a foreign gene
What are the 5 most commonly used expression systems?
E.coli
Yeast
Insect cells/Baculovirus
Mammalian cells
What are 4 advantages of using E.coli as an expression system?
- easy to grow
- inexpensive media
- can produce large quantities quickly (few hours)
- huge quantities if use fermenter chamber
What is the most commonly used promoter to drive expression of our protein?
T7 (phage) promoter
How does the T7 expression system work?
-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
What strain of E. coli is commonly used for plasmid transformation?
BL21 strain
How are E.coli cells that have taken up the plasmid selected for?
Selection using antibodies
What are E.coli cells (as expression systems) grown in, in order to make lots of them?
Shaker culture or in a fermenter
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?
At high quantities
- the optical density is reached when growing in the log/exponential growth phase of the bacterial growth curve
Why would we not introduce IPTG to E.coli expression system to induce T7 promoter expression before reaching the exponential growth phase?
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
