Recombinant Expression Host
Systems
Several host systems are commonly used for
recombinant protein expression
Bacteria (Escherichia coli):
is widely used due to its rapid growth, ease of manipulation, and wellcharacterized genetics. However, it might not be suitable for expressing
complex eukaryotic proteins due to differences in post-translational
modifications.
Yeast (Saccharomyces cerevisiae, Pichia pastoris):
are eukaryotic microorganisms that offer advantages such as the ability
to perform post-translational modifications and secrete proteins. P.
pastoris, in particular, is known for high-level protein expression.
Several host systems are commonly used for
recombinant protein expression
Insect Cells (Sf9, Sf21):
systems like Spodoptera frugiperda (Sf) cells are used to express
proteins that require complex folding and post-translational
modifications typically found in eukaryotic systems.
Mammalian Cells (CHO, HEK293):
are utilized for producing complex proteins that require specific
post-translational modifications, proper protein folding, and
biological activity. Chinese Hamster Ovary (CHO) and Human
Embryonic Kidney (HEK) 293 cells are commonly used.
Expression host systems are employed for the expression
of recombinant proteins both for therapy and research.
PROKARYOTIC EXPRESSION SYSTEMS
E. coli
Prokaryotic expression systems, particularly those
involving bacteria like Escherichia coli, are commonly
used in biotechnology for the production of proteins,
enzymes, and various bioactive compounds.
PROKARYOTIC EXPRESSION SYSTEMS
E. coli
Advantages
• Fast Growth: E. coli grows rapidly and is highly efficient in
replicating DNA and expressing proteins.
• Well-Established: It's extensively studied, and there's a
wealth of knowledge and genetic tools available for
manipulating its genome.
• Cost-Effective: Generally, prokaryotic systems like E. coli are
less expensive to culture compared to mammalian or yeast
systems.
• High Yields: Can produce high levels of recombinant proteins
PROKARYOTIC EXPRESSION SYSTEMS
E. coli
Considerations
✓Lack of Post-Translational Modifications: E. coli lacks the
machinery for complex post-translational modifications like
glycosylation, which is essential for some proteins.
✓Endotoxin Production: E. coli can produce endotoxins, which
might require additional purification steps for proteins intended for
therapeutic use.
✓Protein Folding: Some complex eukaryotic proteins may not fold
correctly or form inclusion bodies in E. coli.
PROKARYOTIC EXPRESSION SYSTEMS
E. coli
Expression Vectors and Systems
Various expression vectors are used in E. coli, allowing for different levels
of protein expression, inducible promoters, and fusion tags to aid in
purification.
Engineering E. coli for Expression
Genetic engineering techniques are employed to optimize expression,
including codon usage, secretion signals, and modification of growth
conditions.
PROKARYOTIC EXPRESSION SYSTEMS
E. coli and other prokaryotic expression systems are suitable for
producing proteins that don’t require extensive post-translational
modifications and are used widely in research, industry, and
pharmaceutical applications.
✓For proteins that need specific eukaryotic modifications or complex
folding, other expression systems like yeast, insect cells, or
mammalian cells might be more appropriate.
✓The choice of expression system often depends on the specific
requirements of the protein, scalability, cost, and intended application
Bacillus subtilis
Advantages
✓Does not produce
LPS/endotoxins.
✓Can be transformed readily
with many bacteriophages
and plasmids.
✓Secrete functional
extracellular proteins directly
into the culture medium.
Considerations
✓Production of
extracellular proteases.
✓Instability of plasmids.
✓Reduced/non expression
of the protein of interest.
Corynebacterium
Corynebacterium species, particularly
Corynebacterium glutamicum, have garnered
attention as potential hosts for heterologous protein
expression and biotechnological applications.
These bacteria have unique metabolic properties,
non pathogenicity and robustness, making them
valuable candidates for expression systems
Corynebacterium
✓Metabolic Versatility: Corynebacteria, notably C. glutamicum, possess a versatile
metabolism capable of utilizing a wide range of carbon sources, which can be
advantageous for producing various metabolites and proteins.
✓GRAS Status: Many Corynebacterium species have Generally Recognized As Safe
(GRAS) status, making them potentially safe for certain biotechnological applications.
✓Secretion Systems: Corynebacteria possess secretion systems that can be harnessed
for the secretion of recombinant proteins, simplifying downstream processing.
✓Tolerance to Harsh Conditions: Some Corynebacterium strains are known for their
resilience in challenging environmental conditions, which can be beneficial in industrial
settings.
✓Genetic Tools: While not as extensively developed as systems like E. coli, there's ongoing
research to improve genetic tools and manipulation techniques for Corynebacterium
species.
Corynebacterium (Application)
✓Production of Amino Acids and Metabolites: Corynebacterium
glutamicum is historically known for its role in the industrial production of amino
acids like glutamate and lysine. Engineered strains are being developed for the
production of other metabolites and bio-based chemicals.
✓Recombinant Protein Expression: Corynebacterium species,
particularly C. glutamicum, are being explored for their potential to express
recombinant proteins. Efforts are ongoing to optimize expression systems for
producing specific proteins of interest.
✓Vaccine Development: Research is being conducted on the use of
Corynebacterium-based systems for vaccine production, utilizing their secretion
systems to express and secrete antigens.
✓Biotechnology and Enzyme Production: These bacteria have been
investigated for the production of enzymes and biochemicals useful in
biotechnological applications.
EUKARYOTIC EXPRESSION SYSTEMS
Eukaryotic expression systems are utilized to
produce proteins, enzymes, antibodies, and other
biologically active molecules within the context of
eukaryotic cells. These systems, derived from
higher organisms like mammals, insects, and
plants, offer advantages for expressing complex
proteins that require specific post-translational
modifications, proper folding, and biological activity
similar to native counterparts
EUKARYOTIC EXPRESSION SYSTEMS
Merits
Drawbacks
• Rapid growth in low cost
medium
• Appropriate posttranslational modifications
• System safety guaranteed
• No endotoxins production
• Hyperglycosylation of
proteins
• Codon bias
• Intracellular retention
Aspergillus niger, Aspergillus oryzae and Trichoderma
Expression platforms for screening and production of
diverse industrial enzymes
Advantages:
Disadvantages:
✓High-level secretion of
enzymes.
✓Large scale production of
recombinant proteins of
eukaryotic origin.
✓Complex
✓Lack of knowledge on
physiology
Yeast expression systems
Saccharomyces cerevisiae
Advantages
Considerations
✓Well-Established System: S. cerevisiae has been
✓Limited Glycosylation: Yeast
extensively studied and is a well-characterized model
organism.
✓High Protein Yield: It can produce high levels of protein.
✓Eukaryotic Post-Translational Modifications: While not
as complex as mammalian systems, S. cerevisiae can
perform some post-translational modifications like
glycosylation and disulfide bond formation.
✓Ease of Genetic Manipulation: It's amenable to genetic
modifications and has a variety of tools available for gene
expression
glycosylation differs from
mammalian cells, which might be
crucial for certain proteins' activity
or stability.
✓Protein Folding: Some proteins
may not fold correctly in yeast,
leading to reduced functionality or
aggregation.
Yeast expression systems
Pichia pastoris
Advantages
✓Strong Promoters: P. pastoris offers strong and tightly regulated promoters,
allowing for precise control over protein expression.
✓High-Level Secretion: Known for its ability to secrete high levels of properly
folded proteins into the culture medium, simplifying downstream purification.
✓Glycosylation: P. pastoris performs simpler forms of glycosylation, which may
be beneficial for some proteins but not as complex as in mammalian cells.
✓Protein Folding: It can properly fold and process certain complex eukaryotic
proteins.
Yeast expression systems
Pichia pastoris
Considerations
Cost of Media: The cost of specialized media for P. pastoris cultures can
be higher compared to E. coli or S. cerevisiae.
Genetic Manipulation: While genetic manipulation is possible, it might
not be as straightforward as with S. cerevisiae.
S. cerevisiae and P. pastoris are widely used in biotechnology and research for the
production of recombinant proteins, enzymes, and various biopharmaceuticals. The
choice between these systems depends on the specific requirements of the protein
being produced, the need for post-translational modifications and scalability.
Insect system
These systems used for production of
✓Recombinant proteins
✓Vaccines
✓Viral pesticides
✓Used in basic research in biology.
✓Cell lines obtained from:
✓Diptera, hemiptera and lepidopteran
insects.
Insect system
High level of expression
Considerations
✓Appropriate posttranslational
modifications.
✓Requires more complex
culture conditions than
bacterial or yeast systems.
✓Less suitable for proteins
needing human-like
glycosylation.
✓Safe for vertebrates
✓Excellent tool for recombinant
glycoprotein production
Mammalian cell expression systems
particularly CHO (Chinese Hamster Ovary)
and HEK293 (Human Embryonic Kidney) cells,
are widely used in biotechnology and
pharmaceutical industries for producing
therapeutic proteins, antibodies, vaccines,
and viral vectors.
Mammalian
CHO (Chinese hamster ovary)
Advantages:
Considerations:
✓Well-established and extensively
used for protein production.
✓Ability to perform complex posttranslational modifications (e.g.,
glycosylation) similar to human
cells.
✓High protein expression levels.
✓Good scalability for large-scale
production.
✓Genetic manipulation can be
challenging.
✓Slower growth rates compared to
bacterial or yeast systems.
✓Requires specific media and culture
conditions.
HEK293 Cells (Human Embryonic Kidney)
Advantages:
Considerations:
✓High transfection efficiency,
allowing for easy introduction of
foreign genes.
✓Ethical considerations due to the
origin of cells.
✓Capable of producing large
quantities of recombinant proteins.
✓Suitable for both transient and
stable expression.
✓Post-translational modification
capabilities.
✓Lower protein production stability
compared to CHO cells.
✓Higher cost associated with culture
and maintenance.
Plant Cells (Nicotiana benthamiana):
Advantage
Considerations
✓ Can produce complex
proteins, antibodies, and
vaccines.
✓ Potential for cost-effective
large-scale production.
✓ Post-translational
modifications, though different
from animal cells, can be
advantageous for specific
applications.
✓ Slower growth rates compared
to some other systems.
✓ Limited availability of
established expression platforms
for certain molecules
Transgenic animal expression systems
Transgenic animal expression systems involve the genetic
modification of animals to produce specific proteins or exhibit desired
traits.
Transgenic Mammals (Goats, Cows, Rabbits): Genetically modified to
produce therapeutic proteins in their milk, such as antibodies, clotting
factors, or hormones. The milk containing the protein of interest can be
harvested and purified for medical use.
Transgenic Mice: Engineered to express genes associated with
human diseases, allowing researchers to study disease mechanisms,
test potential treatments, and understand genetic factors underlying
various conditions.
Conclusion
New generation proteins prepared from the viral/microbial
proteins.
✓Their fragments or the nucleic acid sequences attractive
because of their
✓Stability, non-infectious nature.
✓Homogeneity, cost-effectiveness.
For a specific expression procedure consider
✓Bio-characteristics of the protein
✓Quality
✓Quantity of the protein,
✓Cost
✓Availability
✓Convenience and purposes of the expressed products.
Each system has its advantages and limitations
based on the protein being expressed, desired
post-translational modifications, scalability, and
cost considerations.
The choice of host system depends on the specific
requirements of the protein or molecule being
produced.