SFP15: Experimental Analysis Of Membrane Proteins Flashcards
Name the processes in studying membrane protein structure and function…
1) expression
2) isolation
3) solubilisation
4) purification
5) replacement of pure protein into a native environment
Name 3 examples of high natural sources of proteins..
1) halobacterium — protein: bacteriorhodopsin
2) torpedo California electric organ — protein: acetylcholine receptor
3) bos taurat heart, rich in mitochondria — protein: cytochrome c oxidase
What is Heterologous protein expression?
We get a different cell to make the protein using genetic engineering technologies
What are the problems with prokaryotic expression systems?
- Low yield
- eukaryotic proteins may not express well in bacteria because they dont do the same post translational modifications
- solubility -> improper folding of the protein
What is baculovirus?
Membrane protein expression system- lethal to insects
Produces huge amounts of protein polyhedrin which crystallise in insect gut and kill it
Can alter virus in the lab to replace polyhedrin gene with our gene
Insect cell expression advantages
- ease of manipulation
- high level expression
- membranes
- purification
- scale-up
- insects are eukaryotes
What is the centrifugation step to remove cell debris?
3000 x g 10minutes
Will pellet out any cells that aren’t used
Membranes aren’t heavy enough to be pelleted out
Spins out supernatant
What is the centrifugation step to purify membrane proteins?
100,000 x g 60minutes
Gets a supernatant with soluble protein (not wanted) and you get a cell pellet of cell membranes
What sort of chemicals are good at removing lipids?
detergents
Name 3 classes of detergent
- ionic detergents
- non-ionic detergents
- zwitteronic
what are ionic detergents?
‘Harsh’, can potentially disrupt both hydrophobic forces and ionic forces
Name two examples of ionic detergents
1) Colic acid (similar structure to cholesterol)
2) Sodium dodecyl sulphate (SDS, a C12 fatty acyl chain)
What are non-ionic detergents?
Less ‘harsh’ disrupt hydrophobic forces holding the membrane only
Name three examples of non-ionic detergents
1) ‘triton’ series
2) tween series
3) maltosides
what are zwitteronic detergents?
No net charge, but can disrupt protein:protein and protein:lipid interactions
Give an example of zwitteronic detergent?
Sulfobetaines
What is the chemical difference between a lipid and a detergent?
In a lipid is two hydrophobic chains whereas in a detergent there is only one
Detergents can be water soluble but lipids cannot be
What is the concept of the ‘critical Micelle concentration?’
- At low concentrations detergents are soluble as monomers in water
- Describes the ability of detergents to gang up and form a micelle
- Above the critical micelle concentration (CMC) detergents form micelles
Why can solubilisation lead to loss of activity?
Protein removed from lipid environment necessary for activity
How can some detergents lead to protein denaturation?
Hydrophobic tails of SDS bind tightly to hydrophobic regions of protein, unfold polypeptide, dissociate subunits
What are four methods for reconstitution? (Transfer of proteins from detergent to lipid bilayer)
1) dialysis and dilution
2) gel filtration (size exclusion) chromatography
3) adsorption to polystyrene beads
4) precipitation
What is dialysis and dilution?
- good for detergent with high CMC i.e. where you might not need to remove a very high % of added detergent to trigger the micelle -> bilayer transition
What is gel filtration (size exclusion) chromatography?
- Good for detergents where the micelle is smaller than the lipid-embedded protein
What is adsorption to polystyrene beads?
-Good all round choice as lipids do not interact with polystyrene beads whereas most detergents do
