Flashcards in Lecture 4 Deck (48):
5 areas where protein is localised in Ecoli and features
cytoplasm - largest. Where protein synthesis occurs because it's where ribosomes are.
cytoplasmic membrane - phospholipid bilayer. Cell already has mechanisms to transport proteins here because it does for it's normal transport, membrane and resp proteins
periplasmic space - proteins with disulphide bonds expressed here.
Outer membrane - phospho lip, used to transport proteins
Secreted - completely outside cell in extracellular medium
3 different protein types
cytoplamsic (inner) membrane proteins
outer membrane proteins
properties of soluble proteins
many diff folding structures - but always hydrophobic bit in middle
most found in cytoplasm (cos biggest capacity)
some found on periplasm
properties of cytoplasmic membrane proteins
hydrophobic surface - cos in lipid environment
form alpha helices in transmembrane regions
hard to express at high levels
why are cytoplasmic membrane proteins hard to express at high levels
highly regulated - Because hard to get them to the membrane - most attempts to get them there would result in cell damage so there is very strong regulation
(a reason why you induce protein expression in growth phase - high chance lots will die but hopefully enough around you get a good yield)
properties of outer membrane proteins
form beta barrels -often their function is to provide channels or present proteins on surface
When do inclusion bodies occur
When proteins are expressed in the highest levels in the cytoplasm (biggest capacity), the protein comes together to form inclusion bodies - possibly because hydrophobic proteins can simply clump together without folding
To make inclusion bodies a good way to produce lots of protein for easy purification, what must the proteins be able to do in vitro
be easily refolded
How are proteins freed from inclusion bodies and refolded?
add 6m urea slowly to bodies, proteins will naturally fold
Who's dogma is this method of freeing proteins from inclusion bodies based on and what does it state?
That he protein has all the info it needs to fold correctly encoded in it's own amino acid sequence.
Give an example of a protein which is expressed in inclusion bodies and how it is successfully attained.
Proinsulin - lysozyme added, releases bodies, then given optimum refolding conditions in vitro.
In what form do you need to express a protein if it won't refold itself?
What is the role of chaperone proteins when expressed to improve the solubility of cytoplasmic proteins
help prevent aggregation
Give examples of chaperone proteins
Describe what chaperone proteins do to ensure proper folding
Recognises protein going down wrong route
forms a cavity inside which can accommodate misfolded protein
sets it on it's way again
hopes that next time it will fold right
What catalyses protein folding
as well as chaperone proteins, what helps prevenmt aggregation when proteins are trying to fold?
The masking of the hydrophobic regions
Can the ecoli cytoplasm form disulphide bonds? Why?
No - it's a reducing environment
Where are disulphide containing proteins found? Why?
The periplasm. Because it's oxidising
What's the humidity?
What enzyme catalyses insertion of disulphides?
What enzyme catalyses isomerisation of disulphides?
Describe the common process between both isomeristaion and insertion of disulphides
enzyme sits in oxidised form, acts as an intermediate and is reduced itself.
is formation of disulphides oxidation?
describe basically how the enzymes form the disulphide bonds
by removing the H's from the S on the protein
Give an example in the UK food industry where the presence of DsbA like protein during refolding enhanced activity of recombinant protein
when expressing chymosin
It converts casein to parcasein in curdling so used in cheese production
How many disulphide bonds down chymosin have
What are the 2 ways to make proteins with disulphide bonds
Express proteins in periplasm where disulphides form (call this option 1)
Make cytoplasm more oxidising (option 2)
Describe how you'd go about doing option 1
add signal peptide to the protein - cleaved then just mature protein remains in peri
2 problems with option 1
limited rate of export through the sec machinary - which is what transports proteins to peri
smaller capacity than cytoplasm
2 examples of proteins made using option 1
human growth hormone
antibody fv fragments
2 things you do to achieve option 2
remove thioredoxins and glutaredoxins - (enzymes which make cytoplasm oxidising)
Express DcsB in cytoplasm by removing it's signal peptide so it doesn't go where it normally would
Example of proteins made by option 2 and how many disulphide bonds does it have
human tissue plasminogen activator
Why fuse protein sequences to recombinant proteins
Where can a fusion protein bind to on the recomb protein?
C or N terminus
How is a fusion protein added to a recomb protein?
DNA added into expression vector
Give an example of where a fusion protein has been added to the recomb protein somatostatin, including which end the fusion is added to and where it can be cleaved off
N terminal fusion of Beta-galactosidase.
Can be cleaved in vitro
what is an alternative strategy to adding a fusion protein to increase stability?
Use a protease deficient ecoli strain to incr half life of protein
Another use of fusions, as well as stability
What is the most commonly used fusion tag for purification?
What type of matrix is on the column a protein with a hexa his residue is loaded on? (Once the lysate has been released by breaking open the bacterial cell)
nitrilotriacetic acid matrix (charged with nickel ions)
name the 2 steps undertaken once the His tagged protein has bound to the column
wash - other proteins removed
elute - just his-tagged protein left
Name 2 other fusion proteins and the columns on which they are purified
Glutathione-S-transferase - glutathione affinity column
Maltose binding protein (can target protein to periplasm as well - amylose resin column
What does each fusion protein encode (generally), and what does this permit?
A sequence specific protease cleavage site located in region between affinity tag and cloned gene.
Permits removal of fusion protein tag following affintiy purification (normal purification, like previously mentioned)
What 2 apparatus move recombinant proteins across inner membrane (in secretion)?
Sec or Tat apparatus
What part of the secretory system spans both membranes of ecoli?
Are Ecoli good secretors?
No, they're rubbish. But do have a mechanism to extrete toxins so exploit that