Recombinant Protein Expression - Eukaryotic

Question 1

What are the advantages of using fungal cells as an expression system?

Answer 1

Cheap and easy to grow
Simpler and less expensive than higher eukaryotic systems with usually higher yield
Well-described cell biology and genetics
Very high yields of proteins, even for mammalian membrane proteins
Has been extremely successful in the production of ion channel proteins for structural biology

Question 2

What are the disadvantages of using fungal cells (Pichia pastoris) as an expression system?

Answer 2

Does not support episomal DNA (independent plasmid replication) - requires chromosomal integration of the plasmid DNA
Transformation efficiency can be low (10-100 transformants/microgram of DNA)
Glycosylation may be a problem - not identical to mammalian
For methanol utilizing transformants there is an absolute requirement for oxygen
Potentially very high yields up to 2.5g/l (soluble, secreted), 1.3g/l (cytoplasmic), 1 mg/l (membrane protein)

Question 3

What are the two approaches to driving expression in Sacchromyces cerevisiae (fungal cell)?

Answer 3

Autonomous replication
Two micron based sequence
Typically a marker gene - typically for an enzyme in the yeast cell
Deleted for a enzyme marker - reintroduce it in the plasmid
This forces the yeast cell to select for that marker - auxotrophic marker

Integrative - centromere based sequence, it tricks it into thinking it is a chromosome and is integrated into the genomic DNA
Diploid - two copies
The marker gene is homologous to a marker in the yeast chromosome
By recombination, the cDNA of the protein of interest and promotor is integrated into the chromosome of the host cell
Advantage - permanently producing out gene

Question 4

Describe Pichia pastoria (fungal cell) as an expression system?

Answer 4

AOX1 promotor produces up to 5% total mRNA
A methylotrophic yeast so AOX expression induced by methanol
Yields up to 30% of total cell protein
Genetically similar to Saccharomyces

The AOX1 promotor is repressed by glucose but induced by methanol
As switch from glucose to methanol turns on expression

Question 5

What is involved in a bioreactor of Pichia pastoria uptake?

Answer 5

Stirrer/agitator
Pump and gas mixer for air of N2 and O2
Sparge (gas bubbler)
pH and dissolved oxygen sensor linked to computerised controller
Pumps for feed-in of methanol and base (KOH)

The controller responds to falling pH by increasing input of KOH
In response to falling dissolved oxygen, the controller increases agitator speed and oxygen input

Question 6

What are the two methods for integrating the vector into Pichia pastoria (fungal cell)?

Answer 6

Integration at AOX1
Recombination of the AOX1 promotor with the similar sequence in the chromosomal copy of the AOX1 gene
The recombination writes the expression vector into the chromosomal DNA of the host cell
Advantage is - they contain the full AOX1 gene - so they can still metabolise methanol = mut+
Therefore the expression of the target protein is methanol-inducible

Replacement of AOX1
The plasmid has been linearised
Chromosomal AOX1 is replaced by the plasmid expression cassette
They are fed with glycerine not methanol
No AOX1 oxidase enzyme is produced, cells cannot metabolise methanol, designated ‘mutS’ (for ‘slow’ –some AOX2)
Expression of the ‘target’ protein is methanol-inducible - from AOX2

Question 7

Give a summary of protein expression using fungal cells?

Answer 7

Simple to set up, low costs to operate
Can be done in the lab without specialist equipment, but ‘scale up’ requires a fermenter facility
An ‘industry standard’ for many biopharmaceutical

No major issues with codon usage when expressing mammalian proteins
Yields from Saccharomyces generally on the low side
Yields from Pichia can be very high

Excessive glycosylation of proteins can be a problem

Question 8

Give an overview of insect cells as an expression system?

Answer 8

They are true eukaryotic cells - can produce human cells
They can produce high levels of expression - good at producing membrane proteins and G-coupled receptors
The cell we use are from a fall army worm - they have a natural pathogen
We engineer/culture cells from the gut and infect them with an engineered version of the virus to code for what we want

Question 9

What is the baculovirus and it’s life cycle?

Answer 9

A virus that infects an insect cell
We can use the natural ability of the cell to take over a host environment in order to express or protein

It is a multicapsid polyhedrovirus
The virus is taken up (by a worm) - the alkaline pH in the gut causes the polyhedrin shell to break down
Virus particles can then invade cells in the gut - producing more viruses which bud off and start infecting the whole system
In the later stages each virus particle is repackaged into a polyhedral cell
The cell dies and breaks down allowing the polyhedral cells to be re-released into the environment

Question 10

How is the baculovirus grown?

Answer 10

Tissue culture
This takes place in sterile flasks - fed by culture containing the necessary nutrients and buffer
As cell monolayers in flasks or as suspension cultures in ‘spinner flasks’
Cells may be described as ‘adherent’ or ‘non-adherent’

Adherent cells grow as a monolayer stuck down onto the surface the tissue culture flask
When the monolayer is complete with no gaps remaining it is referred to as a ‘confluent’ culture

Non-adherent cells settle on the bottom of the flask, but are not stuck down
Spinner flasks - allow for much higher cell densities to be achieved

Question 11

What are the steps in expression within an insect cell?

Answer 11

Insert the cDNA for the target protein into a ‘transfer vector’ – a plasmid that can integrate with the rest of the baculovirus genome
Transfect insect cells
Cells will produce recombinant virus that now contains the cDNA for the target protein in its genome
Work out how much virus you have (check the titre)
Then make stocks

Infect cell culture when required for protein expression
Test cells for expression of the protein – continuously monitor to prevent cell death in late infection
Therefore we pick it up at the optimum point in the growth cycle

Question 12

What are some key features of expression using an insect cell as an expression system?

Answer 12

Constitutive expression of the target protein is driven by transcription controlled by the viral polyhedrin promoter
The polyhedrin promoter is highly active in late stage infection
Protein can be secreted from the host cells by making a fusion protein with BiP or the Honey Bee mellitin leader sequence
Baculovirus can be used with Sf9, Sf21 or ‘High Five’ Trichoplusia host cell

Question 13

How is the protein of interest introduced into the baculovirus genome?

Answer 13

The protein of interest encoded into a transfer vector must be combined with a larger baculovirus DNA
Done by either: Double cross over recombination or Transposon-mediated recombination (transposition)

Question 14

Describe double cross-over recombination?

Answer 14

First make a transfer vector - replicated in E.coli - protein of interest is downstream of the promotor and contains orf regions also present in the baculovirus circular DNA
We transfect the insect cells that contain baculovirus DNA with the transfer vector
Double recombination between the two orf sequences - leads to incorporation of the expression cassette
The cells go on to make recombinant baculovirus containing the cDNA of interest
This relies on efficient recombination of the similiar ORF sequences

Question 15

Describe transposon-mediated recombination (transposition)?

Answer 15

Back-to-back system - also relies on a transfer vector - but much more efficient

First make a transfer vector - replicated in E.coli - clone our protein of interest so it is downstream of the promotor and it has two transposable markers Tn7L and Tn7R
These allows recombination with bacmid DNA (essentially a large plasmid containing the baculovirus genome modified to carry lacZ gene)
Bacmid isn’t in the host cell - but in E.coli - therefore this transformation is done in E.coli

Question 16

How do we recover the recombinant baculovirus genome from transposition?

Answer 16

Transposition antibiotic selection - from a LacZ+ E.coli containing recombinant bacmid
Prepare high molecular weight bacmid DNA = recombinant bacmid DNA
Transfect sf9 or sf21 insect cells
This leads to recombinant budded baculovirus particles

Question 17

How/why do we make stocks of the recombinant virus and producing target protein?

Answer 17

From the recombinant baculovirus particles - work out the titre of the virus by viral plague assay = grow a lawn of the insect cells and add a virus solution to see where we have dead cells and which ones were infected
Virus stocks are stored at 4 degrees

When we want to use it we can infect cells for protein expression
We can either make more virus stock or express the protein for experimentation

Question 18

Give a summary of using insect cells as expression systems?

Answer 18

Potentially very high yield of produced protein
Has been very successful in producing mammalian membrane proteins for structural studies, in particular G-protein-coupled receptors
Widely used in industry to express pharmacologically important proteins
Lower cost than mammalian cell culture, but still expensive (doesn’t require any specialised facilities)
No issues with codon usage, folding or post-translational modifications, although glycosylation is ‘non-vertebrate’
Potential for moderate scale-up with suspension culture of cells

Can be technically difficult to set up, but newer systems easier
Long-term preservation of viable virus stocks can be difficult

Question 19

Give an overview of mammalian cells as an expression system?

Answer 19

Small quantities and good functional expression of eukaryotic proteins
They have a native fold and no issues with folding
Expression can be constant (constitutive) or controlled (inducible) by choice of promoter in the plasmid vector.
Promoters generally based on viral (CMV, SV40 MMTV) or mammalian (actin, heat shock protein) promoters that are transcriptionally highly active
Commercially available expression plasmids are ‘promiscuous’ and can be used in many types of cell
The host cell may be a ‘generic’ easy to culture cell (such as HEK293, CHO, COS) or a specific model for the experimental work

But high cost of media and low cell density make it very expensive to produce large quantities of cells

Also cultured on:
This takes place in sterile flasks - fed by culture containing the necessary nutrients and buffer
As cell monolayers in flasks or as suspension cultures in ‘spinner flasks’

Question 20

How do we introduce foreign cDNA into a eukaryote cell in vitro?

Answer 20

Can be injected if the cell is very large - but not practical for many cells

Can use:
Viral vector
Electroporation - expose to a strong electrical field
Lipofection (standard in labs) - combine DNA with a synthetic cation lipid
The DNA lipid complex can be taken up into the cells

Question 21

Describe lipofection?

Answer 21

Liposomes complex with DNA, fuse with the plasma membrane of the cell and enter via endocytosis
They become part of the endosome, the lipid is broken down and the DNA is released to be transcribed within the cell

Question 22

Describe mammalian cell expression vectors?

Answer 22

For propagation use E.coli
β-lactamase gene for ampicillin resistance
Bacterial origin of replication

For selection in cultured mammalian cells:
Neomycin resistance gene driven by viral promoter and polyadenylation sequence

To drive expression of the target protein:
Viral (PCMV) promoter upstream of the sub-cloned cDNA (“gene of interest”) and polyadenylation sequence
Drives the production of stable mRNA

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

Question 23

Why are the elements within the expression vector for a mammalian cell useful?

Answer 23

Cells with stable transfection in which the expression cassette from the vector is integrated into the host cell can be isolated
Integration occurs randomly with very low frequency
Cells with stable transfection isolated by selection for antibiotic resistance
Stably transfected cell lines will be permanently capable of expressing the target protein
But need to be screened in case the integration site is essential for normal cell function and is disrupted by the integration

Question 24

How can site directed recombination within a mammalian cell be carried out?

Answer 24

Using Flp-FRT recombination
Involves the targetted integration to a pre-determined specific site in the host cell at sequences between ‘short flippase recognition target (FRT) sites’ by the recombinase ‘flippase’ (Flp)

A plasmid containing FRT can be integrated with a chromosome also containing FRT, we can have directed/targeted recombination

Integration causes loss of resistance to antibiotic zeocin and gain of resistance to hygromycin (switch antibiotic resistance)
Cells with stable integration selected for growth on hygromycin
The expression cassette is permanently integrated and the new cell line can produce the target protein

Question 25

How can we study gene function further in the expression systems?

Answer 25

Tet on/off system (tetracycline)
We can induce a gene on demand by changing something
A system where expression from a target transgene is dependent on the activity of an inducible transcriptional activator
= control expression

TRE is 7 repeats of a 19 nucleotide tetracycline operator (tetO) sequence - recognised by the tetracycline repressor (tetR)
If tetracycline (or analogue) is present, tetR will bind to tetracycline and not to the TR = allowing transcription

Question 26

What does the Tet- responsive systems require?

Answer 26

It requires transfection with two plasmid vectors:
1. Contains the cDNA for the protein of interest downstream of the TRE-containing promoter, and regions allowing manufacture of the plasmid in E.coli
2. This vector drives expression of the Tet-on or Tet-off transactivator protein (tTA or rtTA), as appropriate
also be manufactured in E.coli, and contains a gene conferring antibiotic resistance for selection of stable mammalian transfectants

Question 27

Describe the Tet on/off system?

Answer 27

Tet-on system
Engineered transcription activator protein (rtTA) binds to tetracycline responsive element (TRE) in the promotor region, upstream of cDNA
This happens only in the presence of tetracycline-type antibiotic (doxycycline)
We can monitor the functional impact on the expression of the protein after we add the tetracycline

Tet-off system
Binding of antibiotic - prevents transactivator (tTA) binding to the TRE
This allows us to look at the expression of protein supporting tumour growth
Suppression of expression with doxycycline limits tumour growth
Co-expression of GFP shows area of target protein expression

Question 28

Describe how we can use viral vectors in mammalian cells for expression?

Answer 28

Recombinant viruses are deficient in some genes, making them incapable of autonomous replication - ‘replication deficient’
The initial expression vector requires co-transfection with ‘helper’ plasmids that encode all the other necessary viral envelope/capsid proteins

Lentivirus - very good for transfecting ‘difficult’ cells including primary cells isolated directly from tissues
Adenovirus gives potentially higher levels of expression - higher concentration (higher titre) of the virus can be made

Question 29

Describe the Lentiviral expression system?

Answer 29

Lenti-X is co-transfected with plasmids encoding viral proteins
Viral proteins are produced and packaged with viral RNA genome
Virus core particle assembled - the genome of interest now codes the ‘protein of interest’
Recombinant viruses bud off from the host cell and can be collected and stored

We now have a virus that will deliver RNA of out viral RNA to a host cell
They are replication deficient viruses - only used for delivery/storage

Question 30

Give a summary of mammalian cells as an expression system?

Answer 30

Highly successful and universally used for functional studies
Scale-up for large scale production of protein prohibitively expensive
A wide range of constitutive and inducible plasmid vector commercially available that can be used in a wide range of cultured cells
Reliable – no codon usage, protein folding or post-translational modification problems
Protein will be targeted to the correct cellular location
Short-term (transient) expression sufficient for most applications
Stable integration of the cDNA of interest can be achieved to produce a cell line permanently capable of expressing the target protein