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Molecular Biotechnology > Lecture 6 > Flashcards

Lecture 6 Flashcards

Mammalian and cell free expression systems

1
Q

When would you use mammalian expression?

A

when exact post translational modifications are essential

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2
Q

Give an example of a ptm which might be required

A

addition of sailic acid in glycans

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3
Q

In what way are mammalian systems similar to insect expression systems

A

They need to exploit viruses

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4
Q

What must a mammalinan expression vector include

A

similar features to yeast and baculovirus/insect cells
strong viral promotor and terminator, splicing signal and polyadenylation signal
viral origin of repl
selectable marker
applicable to virus
Ecoli origin
Ecoli selectable marker
signals for secretion and addition of fusion proteins

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5
Q

Give an example of virus promotor used in mammalian expression systems

A

Simion Virus 40

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6
Q

In what way is eukaryotic promotion different to prokaryotic?

A

IN eukthere is so shine dalgarno, insetead a kozak sequencel.

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7
Q

Give 3 more examples of other viruses used for mammalian expression

A

cytomegalovirus
Rous sarcoma virus
Semlili forest virus

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8
Q

What do the 5’ and 3’ UTR do if added to the gene of interest sequence?

A

increase efficiency of translation

incr mRNA stability

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9
Q

What are 2 common selectable marker in mammalian expression and what does it confer resistance to

A

The neo gene
It encodes resistance to the neomycin
DHFR genes
resistance to methotrexate

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10
Q

What does the DHFR gene normally do

A

The protein it produces (dihydroflorate reductase) reduces dihydrofolate to tetrahydrofolate.
It’s essential for mammalian purine synthesis

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11
Q

How does Methotrexate act to inhibit DHFR

A

binds to it’s active site, acts as competitive inhibitor

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12
Q

How can MTX sensitivity be overcome

A

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.

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13
Q

So what is the high conc of MTX acting as?

A

selection pressure

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14
Q

What 3 kinds of mammalian cells are often used for mammalian expression

A

green monkey kidney
baby hamster kidney
human embryonic kidney

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15
Q

How is DNA introduced into mamm cell cytosol?

6 ways

A 
microinjection
electroporation
calcium phosphate 
cationic lipids
liposome mediated
viral vector
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16
Q

To be transcribed where must the desired gene go once in the cytosol?

A

Nucleaus

17
Q

How does the gene get to where it needs to be transcribed?

A

nuclear targetting sequences are added to it

18
Q

what must be created for long term production of a recombinant protein? And define it.

A

stable cell line

=where plasmid has been transfected into cell line then INCORPORATED INTO GENOME.

19
Q

what kind of cells are often used to make stable cell lines

A

chinese hamster ovary cells

20
Q

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

A

Human DNase1
Treats CF
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
decr antigenicity

21
Q

Why are cell free expression systems plausible?

A

Don;t need cell integrity for protein synthesis - cell extracts contain all machinary and biochem constituents required for scription and lation.

22
Q

what are the 2 main benefits of cell free expression

A

prevents toxicity problems

makes membrane protein expression easier

23
Q

Describe a cell free expression system

A

continuous exchange system
2 halves seperated by semi permeable membrane
Top half - reaction chamber all major components
Dna template
T7 RNA pol
Ecoli translation machinary
energy regenerating system (gg cos otherwise lots of ATP used)

bottom half - feeding chamber
amino acids
buffer componenents
NTPs
energy substrates
24
Q

How do cell free expression systems work

A

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

25
Q

What’s are the 3 main benefist of cell free

A

you only translate what’s on vector so purifying is easier
easier to manipulate conditions
easier to purify UNTAGGED protein after

26
Q

What yield can ecoli lysates give as cell free systems?

A

> 1mg/ml

27
Q

What’s added to help synthesis certain types fo proteins cell free way?

A

additional factors

28
Q

give 2 broad examples of successful recomb proteins from ecoli lysate

A

Eukaryotic protein with multiple disulphide bonds
Multimeric co factor containing protein synthesised
-added range of ecoli chaperones
-cofactor added directly to rxn mix

29
Q

What’s an alternative to ecoli lystae as cell free expression system?

A

wheat germ

30
Q

why are wheat embryos ie seeds very promising for cell free expression

A

have preassembled DNA and RNA pol and other components for scription and lation

31
Q

What are the positives of a wheat germ cell free system

A

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)

32
Q

Main disadvantage of wheat germ line cell free expression

A

No glycozza.

33
Q

why are cell free expression systems just so good for membrane proteins

A

due to toxicity in cellular expression systems

most things you try to attach protein to membrane of die

34
Q

How are membrane proteins extracted from cell free systems - 3 ways

A

Detergent e.g. DMM added, membrane protein directky inserts into micelles.
liposomes
protein nanoids

Molecular Biotechnology (14 decks)

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