Introduction to different host systems for protein expression. Advantages and disadvantages of different host systems

1. Model Host Systems
Choice of host depends on several needs:
 Costs and availability of supports (kits, vectors, reagents and expertise)
 Yield – large mass of protein presents specific challenges for scale up (bacteria/yeast vs.
mammalian)
 How will the protein be used after expression?
 Toxicity of protein on host cell
 Post-translational modification
 Impact on host cell on protein solubility and hydrolysis
 Location of protein within the host cell – membrane, inclusion body, secretion into media
 Complexity of medium
(A) E. coli
Advantages:
 First choice due to level of support (technical, literature, kits, plasmids, reagents)
 High yield and easier scale up (up to 100 mg per liter of culture)
 Cost of media and reagents is relatively inexpensive
 Many expression plasmids – many with gene expression regulation
 Fast growth condition – one to two days from starter culture to cell pellet
 Up to 50% of protein can be target protein
 Simple culture conditions
 Simple transformation protocols
 Many parameters can be altered to optimize expression
Disadvantages:
 Minimal post-translational modification
 Lipid modification, phosphorylation, glycosylation, decarboxylation, acetylation
can not be performed, these may be critical requirements for functional protein
 Large proteins may be difficult to express (plasmid size is limited to about 15 kb)
 Poor folding of proteins in the cytoplasm
 In vitro refolding protocols are inefficient in this system

2.  Large number of proteins are toxic to E.coli
 Inclusion body packing of protein
 Insoluble, aggregated and non-native protein
 Often times takes place in high expressed or toxic proteins
 Codon Use differences, false stops and low expression
 Inefficient disulfide bond formation
 Endotoxin production
Example of expression vector used: pET series, pGEX series, pBAD
(B) Fungi
(I) Saccharomyces cerevisae
Advantages:
 Good expression levels
 Choice of secreted or cellular expression
 Low cost
 Simple culture conditions
 Scalable
 Able to perform most eukaryotic post-translational modifications
 Efficient protein folding
 Endotoxin free
Disadvantages:
 Likely lower expression than Pichia pastoris
 Secretion likely lower than Pichia pastoris
 Glycosylation still different to mammalian cells
 A tendency to hyperglycosylate proteins
 N-glycan structures considered allergenic

3. (II) Pichia pastoris
Advantages:
 High expression levels
 Low cost
 Simple culture conditions
 Relatively rapid growth
 Scalable
 Choice of secreted or intracellular expression
 Protein secretion efficient and allows simple purification
 Extensive post-translational modification of proteins
 Efficient protein folding
 N-glycosylation more like higher eukaryotes than with Saccharomyces cerevisiae
 Endotoxin free
Disadvantages:
 Use of methanol as inducer is a safety (fire) hazard at scale
 Glycosylation still different to mammalian cells
Example of expression vector used: pAO815, pGAPZ, pPIC series, pHIL series
(C) Mammalian cell lines
Advantages:
 Good expression levels
 Moderately scalable
 Suspension-adapted cells facilitate scale-up
 Efficient protein folding
 Good for secreted proteins
 All post-translational modifications
 Endotoxin free
Disadvantages:
 Expensive culture media
 Complex growth requirements

4. Example of expression vector used: pcDNA series, pCMV series, pJRed series
(D) Insect cells (Baculovirus infected insect cells)
Advantages:
 Good expression levels (especially for intracellular proteins)
 Relatively rapid growth
 Efficient protein folding
 Moderately scalable
 Extensive post-translational modification of proteins
 Glycosylation more like mammalian cells
 Relatively deglycosylate protein enzymatically (good for structure determination)
 Endotoxin free
Disadvantages:
 Expensive culture media
 Large scale of virus needed on scale-up
 Inefficient processing of pro-peptides in the secretory pathway
 Glycosylation still different to mammalian cells
 Viral infection leads to cell lysis and potential degradation of expressed proteins
Example of expression vector used: pAcP series, pBAC series, pAcUW31
(E) Transgenic plants:
Advantages:
 Plants contain no known human pathogens (such as prions, virions, etc.) that could
contaminate the final product
 Higher plants generally synthesize proteins from eukaryotes with correct folding,
glycosylation & activity
 Significantly lower production cost than with transgenic animals, fermentation or
bioreactors
 Reducing the costs of storage and transportation of recombinant proteins (when they are
produced in dry textures like grains)

5.  Low ethical concerns
 Easier purification (homologs don’t pose any purification challenge, e.g. serum proteins
or antibodies)
 Versatile (production of a broad diversity of proteins)
Disadvantages:
 Take more time to develop
 Transgene & protein pollution
(F) Transgenic animals:
Advantages:
 Low cost of production
 More efficient system
 Higher stability of proteins
 All the post-translational modifications
 Reduced risk of immunogenicity
 No or reduced aseptic conditions required
Disadvantages:
 Limited by legal and ethical restrictions
 Health related complications in transgenic animals
 Low rate of success
 Prior extensive knowledge required
 Potential pathogenic infection
 Relatively time consuming
 Scaling up problems