Recombinant Protein Expression 1 & 2

Question 1

Give some examples of possible expression systems?

Answer 1

In vitro, prokaryotic, yeast cells, fungal cells, insect cells, mammalian cells.

Question 2

Which expression systems are for quality over quantity?

Answer 2

Fungal cells, insect cells and mammalian cells.

They produce small quantities but have good functional expression of eukaryotic proteins, usually have a native fold and post-translational modification.

Limited number, but increasing.

Question 3

Which expression systems are for quantity over quality?

Answer 3

Prokaryotic, yeast cells and fungal cells.

Produce large quantities but may have poor expression of eukaryotic proteins, problems with solubility of multi domain proteins, little post translational modifications.

Many.

Question 4

What are the advantages of using E.coli as a host for recombinant protein expression?

Answer 4

Simple and rapid to culture, easy to transform, well characterised genome sequence, range of vectors/markers.

Question 5

What are the disadvantages of using E.coli as a host for recombinant protein expression?

Answer 5

Requires cDNA (no introns), lacks much post-translational processing, possible protein stability/solubility/toxicity issues.

Question 6

How do you opsonise codon usage?

Answer 6

Some E.coli have additional tRNA genes to enhance expression of these genes, mutate critical codons to more commonly used codons or resynthesise the complete gene to reflect host codon usage.

Does not change encoded protein sequence.

Question 7

What do fusion tags allow?

Answer 7

Easy identification and purification of protein, some can improve stability and folding.

Question 8

What are fusion tags?

Answer 8

Independent of host organism, they can be added to either N- or C- terminal and in-frame with the protein coding region. PCR is usually used to amplify the correct coding region.

Question 9

What is affinity chromatography used for?

Answer 9

To purify tagged proteins

Question 10

How does affinity chromatography work?

Answer 10

Involves the specific binding of a protein to a ligand immobilised onto a support matrix. Non-tagged protein pass through and any that stick non-specifically are washed off before the recombinant protein is specifically eluted

Question 11

What are some common fusion tags and their columns for affinity chromatography?

Answer 11

Glutathione-S-transferase and glutathione, maltose binding protein and amylose, hexa-histidine tags and metals (eg. Nickel/Cobalt), streptag 2 and streptactin.

Question 12

How are streptag usually added?

Answer 12

Genetically fused to the protein to be purified during cloning into the expression vector. By removing the stop codon and then genetically fusing in-frame.

Question 13

What are the advantages of expressing in yeast cells?

Answer 13

Flexible, can have high or low copy number plasmids, inducible or constitutive promoters are available. Some prokaryotic post-translational modifications. Deletions of genes of homologous proteins allows functional assays in vivo by complication. Multiple plasmids can be obtained therefore multiple proteins can be expressed, cheap and easy to grow. Have well derive cell biology and genetics.

Question 14

Define a recombinant protein?

Answer 14

A protein made in a non-native cell using a ‘gene’ constructed in the lab via recombinant DNA technology.

Question 15

Define the term expression?

Answer 15

The process of transcription and translation resulting in production of a recombinant protein.

Question 16

Define vector?

Answer 16

Any plasmid or virus used to carry a cDNA encoding the protein of interest into the cell that you want to express it.

Question 17

Define the term cloning/sub cloning?

Answer 17

The process of integrating the cDNA of interest into the expression vector.

Question 18

Define the term host cell?

Answer 18

The cell in which you are trying to express the protein (the cell ‘hosts’ the vector).

Question 19

Define the term transfection?

Answer 19

The process of introducing a vector into a cell.

Question 20

What do you need to know about the protein prior to choosing an expression system?

Answer 20

Is it prokaryotic or eukaryotic?
Is it soluble or membrane-associated?
Is it post-translationally modified and does this effect it for your studies?
What will it be used for and how much will you require?

Question 21

Define the term induction?

Answer 21

The process of switching on transcription from the vector once it’s in the cell.

Question 22

Define constitutive expression?

Answer 22

Expression where RNA transcription is driven by a promoter that is permanently active (such as the cytomegalovirus (CMV) promoter).

Question 23

Define inducible expression?

Answer 23

Expression where RNA transcription is driven by a promoter that is switched on by hormone/drug/metabolite-dependent binding of a trans activator.

Question 24

Define transient expression?

Answer 24

Protein expression over a short period (1-3 days) under conditions where there is no selection for the vector in the expressing cell.

Question 25

Define stable expression?

Answer 25

Protein expression by the host cell that is permanent – the vector DNA can be integrated into the host cell genome. This is detected by screening for co-integration of an antibiotic resistance gene.

Question 26

How are higher eukaryotic cell grown?

Answer 26

By tissue culture in sterile flask fed by culture containing all the required nutrients and buffered by bicarbonate.

Question 27

What’s the difference between adherent or non-adherent?

Answer 27

Adherent cells grow as a mono layer stuck down onto the surface of the tissue culture flask, when complete it is referred to as a confluent culture.
Non-adherent cells settle on the bottom of the flask but are not stuck down.

Question 28

Why so mono layer culture a problem and what other method can be used?

Answer 28

Only produced a few million cells and scaling it up is expensive. Suspension cultures in spinner flasks allows for higher densities to be achieved.

Question 29

What are the basic steps when selecting an expression system?

Answer 29

1) Clone a cDNA for your target protein
2) modify so that it codes for segments that allow purification or detection
3) select an appropriate expression system to test, insert your cDNA into it
4) transform or transfect cells
5) induce cells and test for the presence of your protein.

Question 30

What methods are used to get foreign cDNA into a eukaryotic cell cultured in vivo?

Answer 30

Direct injection into the nucleus, viral transfection, electroporation, lipoinfection

Question 31

When is direct injection of DNA into the nucleus suitable?

Answer 31

For driving expression in a small number of very large cells.

Eg. Oocytes of the frog xenopus

Question 32

What method of introduction of DNA into cells has the highest transfection efficiency?

Answer 32

Viral transfection

Question 33

Describe lipoinfection?

Answer 33

Lipid-DNA complexes are adsorbed onto the surface of the cell and taken up by endocytosis. Fusion of the liposome and endosomal membrane releases the vector DNA from the complex. Medium transfection efficiency.

Question 34

Describe mammalian expression systems?

Answer 34

High level expression, but expensive to produce large quantities of cells.

Question 35

What type of promoters do mammalian expression systems have?

Answer 35

Constitutive or inducible promoters based on viral (CMV, SV40 MMTV) or mammalian (actin, heat shock protein) promoters.

Question 36

Give some examples of mammalian expression systems?

Answer 36

‘T-rex’ Tet-on/off systems (Invitrogen)- tetracycline inducible
Ecdysone (an insect hormone)-inducible systems (Invitrogen)
MMTV promoter system (Pharmacia)- induced by glucocorticoid (dexamethasone).
CMV/lac hybrids (Stratagene)- Repressor lacI from cotransfected plasmid represses expression. Inducible by IPTG.

Question 37

How are stable transformants of mammalian expression systems selected?

Answer 37

By antibiotic resistance

Question 38

What features does a mammalian cell expression vectors have?

Answer 38

Bacterial ori and ß-lactamase gene for ampicillin resistance for propagation in E.coli cells. Neomycin resistance gene driven by a viral promoter and poly adenylation sequence for selection in cultured mammalian cells. Viral promoter upstream of inserted cDNA of interest and poly adenylation sequence to drive expression.

Question 39

What may the promoter be in a mammalian expression vector?

Answer 39

Promoter may be constitutive, or inducible with hormones (ecdysone), drugs (dexamethasone), metabolites (IPTG)

Question 40

Describe the tet system?

Answer 40

Allow expression to be either switched on or off at defined times by addition of tetracycline-like drugs to the culture medium.
The system has two key parts: When host cells are transfected with a ‘tet-on’ or ‘tet-off’ plasmid, transcription of the cDNA encoding the target protein (‘gene of interest’) is driven by an engineered CMV promoter that also contains ‘tetracycline responsive elements’. TREs bind a transactivator protein – in the tet-on system, binding of the transactivator to the promoter only occurs when the tetracycline-like drug doxycycline is present. Thus addition of doxycycline to the cell culture medium will switch on expression. In the tet-off system, the transactivator has been modified so that it normally binds to the TRE of the promoter, so transcription is normally active. In the tet-off system, binding of doxycycline to the transactivator protein PREVENTS it from binding to the promoter of the expression vector, so addition of the drug to the culture medium switches off expression.

Question 41

When is the tet-off system useful?

Answer 41

If the researcher wants to see to what extent a biological phenomenon is dependent on the presence of a particular protein.

Question 42

How are insect eukaryotic expression systems infected with DNA of interest?

Answer 42

By exploiting the life cycle of the baculoviruses.

Question 43

What is the transfer vector in baculoviruses expression system?

Answer 43

The vector in which the cDNA of interest is sub-cloned. Does not encode any parts of the baculovirus particle, but is engineered so that it can recombine with the second part of the vector that does.

Question 44

Where is the second part of the plasmid in the baculoviruses expression system stored?

Answer 44

In specially-engineered E.coli cells.

Question 45

When both parts of the baculoviruses expression system vector are together what is formed?

Answer 45

A bacmid DNA that encodes all the components of the baculovirus and the protein you are trying to express.

Question 46

What are the steps of the expression process in baculoviruses?

Answer 46

1) Recover high molecular mass ‘bacmid’ DNA from E.coli, and check for insert by PCR.
2) Transfect insect cells. Recover recombinant virus, check titre, make stocks.
3) Transfect cells as required for expression.
4) Constitutive expression from viral polyhedrin promoter
5) Protein can be secreted by making fusion with BiP or Honey Bee mellitin leader sequence.
6) Baculovirus can be used with Sf9, Sf21 or ‘High Five’ Trichoplusia cells (reported to give much higher expression and to grow faster).

Question 47

How are transfer vectors made for the baculoviruses expression system?

Answer 47

This is done by either double cross-over recombination or transposon-mediated recombination (‘transposition’).

Question 48

What are the components of a baculoviruses expression system?

Answer 48

For replication in E. coli: 
LacZ for Blue/white selection of clones, Phage f1 region for single stranded DNA recovery, β-lactamase gene confers resistance to ampicillin and Ori. 
For integration (‘bacmid’ DNA):
Transposable elements drive recombination (Tn7L&R), Gentamycin resistance gene for selection of transfected cells, Promoter for polyhedrin viral coat protein, Multiple cloning site for insertion of cDNA for protein of interest, Polyadenylation sequence for mRNA.

Question 49

What are replication deficient virus vectors?

Answer 49

Recombinant viruses are deficient in some genes, making them incapable of autonomous replication.

Question 50

What are lentiviruses good for?

Answer 50

Transfecting ‘difficult’ cells including primary cells isolated directly from tissues

Question 51

What can adenoviruses potentially to better than lentiviruses?

Answer 51

Higher levels of expression- higher titre virus can be made.

Question 52

What is the lentivirus structure?

Answer 52

The RNA and virally-encoded enzymes (polymerase, reverse transcriptase, RNase, integrase) are packaged into core shell (p24 protein). Surrounded by a membrane envelope that overlies a layer of matrix protein and contains transmembrane glycoproteins gp41 and gp120.