Flashcards in Lecture 6 Deck (34):
When would you use mammalian expression?
when exact post translational modifications are essential
Give an example of a ptm which might be required
addition of sailic acid in glycans
In what way are mammalian systems similar to insect expression systems
They need to exploit viruses
What must a mammalinan expression vector include
similar features to yeast and baculovirus/insect cells
strong viral promotor and terminator, splicing signal and polyadenylation signal
viral origin of repl
applicable to virus
Ecoli selectable marker
signals for secretion and addition of fusion proteins
Give an example of virus promotor used in mammalian expression systems
Simion Virus 40
In what way is eukaryotic promotion different to prokaryotic?
IN eukthere is so shine dalgarno, insetead a kozak sequencel.
Give 3 more examples of other viruses used for mammalian expression
Rous sarcoma virus
Semlili forest virus
What do the 5' and 3' UTR do if added to the gene of interest sequence?
increase efficiency of translation
incr mRNA stability
What are 2 common selectable marker in mammalian expression and what does it confer resistance to
The neo gene
It encodes resistance to the neomycin
resistance to methotrexate
What does the DHFR gene normally do
The protein it produces (dihydroflorate reductase) reduces dihydrofolate to tetrahydrofolate.
It's essential for mammalian purine synthesis
How does Methotrexate act to inhibit DHFR
binds to it's active site, acts as competitive inhibitor
How can MTX sensitivity be overcome
put more copies of DHFR gene in.
Do this via breeding strain to have more copies of the plasmid present by slowly incr conc of MTX.
So what is the high conc of MTX acting as?
What 3 kinds of mammalian cells are often used for mammalian expression
green monkey kidney
baby hamster kidney
human embryonic kidney
How is DNA introduced into mamm cell cytosol?
To be transcribed where must the desired gene go once in the cytosol?
How does the gene get to where it needs to be transcribed?
nuclear targetting sequences are added to it
what must be created for long term production of a recombinant protein? And define it.
stable cell line
=where plasmid has been transfected into cell line then INCORPORATED INTO GENOME.
what kind of cells are often used to make stable cell lines
chinese hamster ovary cells
Give 2 examples and details of recombinant proteins made in mammalian cells used in human therapeutics, including how the gene's efficiency has been increased
Is a 37kDa secreted glycoprotein with 2-N glycosylation sites and 2 disulphide bonds
sialic acid included in glycans
Site directed mutagenesis has altered it's structure so it is not inhibited by actin (which is released in lung when cells die)
Factor VIII -- blood clotting for haemophilia
Is a precursor to factor VIIIa which catalyses activation of factor IX to IXa
currently made in CHO - ££ to synthesis and purify
Icr resistance to inactivation - incr time before degraded
incr efficiency of activation
Why are cell free expression systems plausible?
Don;t need cell integrity for protein synthesis - cell extracts contain all machinary and biochem constituents required for scription and lation.
what are the 2 main benefits of cell free expression
prevents toxicity problems
makes membrane protein expression easier
Describe a cell free expression system
continuous exchange system
2 halves seperated by semi permeable membrane
Top half - reaction chamber all major components
T7 RNA pol
Ecoli translation machinary
energy regenerating system (gg cos otherwise lots of ATP used)
bottom half - feeding chamber
How do cell free expression systems work
bottom half contains 'ingredients'. When ingredients at low conc in top they move up through semi permeable membrane by diffusion. Mean while inhibitory by-products move down
What's are the 3 main benefist of cell free
you only translate what's on vector so purifying is easier
easier to manipulate conditions
easier to purify UNTAGGED protein after
What yield can ecoli lysates give as cell free systems?
What's added to help synthesis certain types fo proteins cell free way?
give 2 broad examples of successful recomb proteins from ecoli lysate
Eukaryotic protein with multiple disulphide bonds
Multimeric co factor containing protein synthesised
-added range of ecoli chaperones
-cofactor added directly to rxn mix
What's an alternative to ecoli lystae as cell free expression system?
why are wheat embryos ie seeds very promising for cell free expression
have preassembled DNA and RNA pol and other components for scription and lation
What are the positives of a wheat germ cell free system
seeds are cheap and safe
easy to scale up
designed so any cDNA can be PCR amplified to contain correct 5' and 3' UTRs.
PCR product alone can be used in this system
can use in continuous exchange
doesn't have strong codon preference (unlike ecoli)
Main disadvantage of wheat germ line cell free expression
why are cell free expression systems just so good for membrane proteins
due to toxicity in cellular expression systems
most things you try to attach protein to membrane of die