7: Sources of recombinant proteins Flashcards
What are the advantages and disadvantages of bacterial expression, and give an example.
Advantages:
- Well characterised
- High level of protein expression (30% of total cellular protein)
- Rapid
- Cheap
Disadvantages:
- Different proteins accumulate intracellulary (complicated downstream processing homogenisation, removal of cellular debris and extensive purification)
- No post-translational modifications (glycosylation)
- Presence of lipopolysaccharides on the surface (pyrogenic)
Example: Interleukin-2 (IL-2)
A T-cell growth factor produced by T-cells in response to activation by antigen. It activates many aspects of immune response. It is a glycoprotein consisting of a single chain polypeptide of 133 amino acids. The unglycosylated form displays the same biological activity to native glycosylated form.
Choosing an appropriate expression system is dependent on?
- Costs
- Speed
- Folding
- Safety
- Scalability
- Post-translational modification
What are the advantages and disadvantages of yeast expression and give an example.
Advantages:
- Well characterised
- Generally regarded as safe
- Grow relatively quickly in an inexpensive media
- Tough outerwall protects it against physical damage
- Industrial scale fermentation
- Post-translational modification
Disadvantages:
- Glycosylation pattern differs from native protein (short half life and triggers rapid clearance from blood)
- Some yeast motifs can be immunogenic
- Expression levels low (5% of total cellular protein)
Example: Gardasil
Cervical cancer vaccine approved for the prevention of cervical, vulvar and vaginal cancers caused by HPV types 16 + 18
What are the advantages and disadvantages of insect cell (Baculovirus) expression and give an example.
Advantages:
- High level of intracellular protein expression
- Rapid and inexpensive culture
- Free of human pathogens
Disadvantages:
- Targetted intracellular production (Low level of accumulation of desired protein (mg/L range)
- Post-translational modifications can be incomplete and vary from native form (especially glycosylation)
Example: Cervarix
Cervical cancer vaccine approved for the prevention of cervical, vulvar and vaginal cancers caused by HPV types 16 + 18. It is produced by baculovirus expression system using cells derived from the insect Trichoplusia Ni
What are the advantages and disadvantages of mammalian cell expression and give a few examples.
Advantages:
-Post-translational modifications
Disadvantages:
- Complex nutritional requirements
- Slow growth
- Susceptible to physical change
- Increased production costs
Examples:
SV40 - Plasmid replication in host
CMV promoter (promiscuous) - constitutive expression of insert
MCS - unique restriction sites for cloning
Ampr - ampicillin resistance
Neo (neomycin phosphotransferase gene) - resistance to antibiot G-418
Explain the transformation/transfection of mammalian cells
- Calcium/phosphate form a precipitate with DNA. This precipitate complexes taken into the cell by endocytosis.
- Lipofection is a cationic lipid based system that forms micelles with DNA inside that fuses with the plasma membrane
- Electroporation: subjects cells to short but high energy electric pulses that briefly open the cell membrane, driving the charged DNA into cells.
What are the advantages and disadvantages of plant expression and give an example.
Advantages:
- Low cost of plant cultivation
- Harvest methodologies inexpensive and well established
- Ease of scale up
- Proteins expressed in seeds stable in seed for a long time
- Free of human pathogens
Disadvantages:
- Variable/low expression levels
- Potential of post-transcriptional gene silencing (Sequence specific mRNA degradation)
- Glycosylation patterns differ from native human products (Plant glycoforms immunogenic)
- Environmental/public concerns (potential escape of genetically altered plants)
- Seasonal/geographical nature of plant growth.
Example: A plantibody produced in tobacco has been approved for use in europe, it binds specifically to streptococcus mutans (a bacteria that cause tooth decay) It prevents the bacteria from adhering to teeth and can effectivily eliminates S.mutans for up to 2 years.
What are the advantages and disadvantages of transgenic animal expression and give an example.
Advantages:
- Ease of harvesting crude product
- Availability of commercial milking system designed to maximise process hygiene
- High expression levels (1-60g/L)
- Ongoing supply of product
- Guaranteed by breeding
- Milk proteins are well characterised (rational development of downstream processing protocols)
Disadvantages:
- Variability of expression levels
- Characterisation of exact nature of post-translational modifications (Glycosylation of tPA differs from murine cell culture systems)
- Lag time between generation of transgenic embryo and commencement of routine product manufacture.
Example: Atryn (anti-thrombin)
What are master and working cell banks?
Master and working cell banks are product producing cell line aliquoted and stored in liquid nitrogen master cell bank (>200 ampoules). The masterbank ampoule is expanded. Single ampoule -> Lab scale starter culture (200-300mL) -> Small bioreactor (Couple of L’s) -> Production scale bioreactor (1000’s of L)
