The document discusses mammalian cell expression systems, which are essential for producing proteins with proper folding and post-translational modifications crucial for biological activity. It outlines the advantages and applications of these systems, such as producing monoclonal antibodies and therapeutic proteins, particularly emphasizing the roles of various cell lines like HEK 293 and CHO. Additionally, it highlights challenges such as high costs and contamination risks that impact large-scale usage.

1. MAMMALIAN CELL EXPRESSION
SYSTEM, STRONG PROMOTERS
MADE BY- SARAH HAMID
M.Sc BIOTECHNOLOGY
3rd SEMESTER
SUBJECT- ANIMAL BIOTECHNOLOGY

2. INTRODUCTION
• Expression can be described as a transcription and translation of a
gene. A particular host is required to express a particular gene.
• The production of proteins in appropriate quantity and quality is an
essential requirement of the present time. There appears to be a
progressive increase in the application of mammalian cells for
proteins production.
• Expression systems utilizing mammalian cells for recombinant
proteins are able to introduce proper protein folding, post-
translational modifications, and product assembly, which are
important for complete biological activity.
• Today, there are wide selections of expression systems available
for large-scale recombinant protein production. These expression
systems include E. coli, baculovirus-mediated insect cell expression,
yeast, and several mammalian based systems.

3. WHAT IS A MAMMALIAN CELL EXPRESSION
SYSTEM?
• Mammalian expression is the system of choice for studying
the function of a particular protein in the most
physiologically relevant environment, because it allows for
the highest level of posttranslational processing and
functional activity of the protein.
• It is commonly used for the production of antibodies and
therapeutic proteins, as well as for proteins that will be used
for human use in functional cell-based assays.

4. • A number of mammalian cell lines have been utilized for protein
expression with the most common being HEK 293 (Human
embryonic kidney) and CHO (Chinese hamster ovary). These cell
lines can be transfected using polyethyleneimine (PEI) or calcium
phosphate. HEK 293 cells exhibit the highest level of PEI-
mediated transfection with 50–80% of cells showing green
fluorescent protein (GFP) expression, and are now widely used for
the production of recombinant proteins both by transient
transfection as well as by the formation of stable cell lines.
• Protein expression in mammalian cells can also be achieved using
viral-mediated transduction by such techniques as the BacMam
system. This technology utilizes recombinant baculoviruses for
simple transduction of mammalian cells, allowing for the
production of milligram quantities of protein for structural
studies.

5. FACTORS FOR SELECTING AN EXPRESSION SYSTEM
S.No Factors effecting gene expression
1 Facilities of the laboratory and local expertise.
2 Type of protein to be expressed.
3 Whether the protein is toxic?
4 Whether carbohydrate or other modification required?
5 Reqiurement of large yield of protein.
6 How to purify protein?
7 Production costs.
8 Regulatory considerations.
9 Safety considerations.
10 Purpose for which it is required.

6. MAMMALIAN CELL-BASED EXPRESSION
SYSTEM COMPARED

7. REQUIREMENTS FOR MAMMALIAN CELL
EXPRESSION SYSTEM
(1) VECTORS - Vectors are autonomously replicating DNA molecules that can be
used to carry foreign DNA fragments. It is a vehicle used in gene cloning.
DNA of interest is first cloned into an appropriate vector and then by
transfection, the gene can be inserted into the host for its expression. For
expressing heterologous genes in mammalian cells, usually vectors derived
from mammalian viruses are used. These include viruses such as Simian
Viruses 40 (SV40), polyomavirus, herpesvirus. In order to construct vector
the requirement is to select an efficient promoter and also the selection
marker.
(2) PROCESS FOR TRANSFER OF GENE - There are two general methods for the
introduction of foreign DNA into mammalian cells. One is mediated by virus
infection and the other by direct transfer of DNA into the cells employing
chemical liposomes, calcium phosphate, DEAE-de-xtran and polybrene and
physical electroporation and microinjection methods.Calcium-phosphate, PEI
and electroporation have been shown to be useful as vehicles/approaches
for large-scale transient gene expression.

8. REQUIREMENTS FOR MAMMALIAN
EXPRESSION SYSTEM
• The expression level of a gene largely depends upon how
efficiently it is transcribed.
• Transcription of any gene takes place when the RNA polymerase
complex interacts with the promoter sequences and moves along
the gene from a 5ı to 3ı direction.
• This produces an RNA transcript and finally dissociates from the
gene at the transcription signal, freezing the transcript for
eventual translation.
• The gene expression in mammalian cells needs a suitable cell line
and the appropriate vectors that should act as a vehicle to
transport the gene of required interest into the required cell
lines.

9. WHAT IS THE ADVANTAGE OF MAMMALIAN
CELL EXPRESSION SYSTEM?
• The main advantage of mammalian cell expression is that the
mammalian cells can properly and efficiently recognize the
signals for synthesis, processing and secretion of eukaryotic
proteins.
• Therefore expression systems utilizing mammalian cells for
recombinant protein manufacture are able to introduce proper
protein folding, post-translational modifications, and protein
assembly, which are important for complete biological activity.
• From the pharmaceutical field, to almost all biopharmaceutical
companies in the world rely on the use of mammalian-cell-based
stable cell lines for the manufacture of biologics.
• The introduction of large-scale transient transfection has
enabled the use of mammalian cells more attractive in terms of
speed and ease of use, particularly for cell surface and secreted
glycoproteins.

10. ANTIBODY EXPRESSION IN THE MAMMALIAN CELL SYSTEM

11. APPLICATIONS OF MAMMALIAN CELL
EXPRESSION SYSTEM
• Production of monoclonal antibody- Mammalian cells are currently the
main hosts for the commercial production of therapeutic proteins, including
monoclonal antibodies (mAbs).African green monkey kidney (COS) cells may
be appropriate if the aim is to produce small-scale quantities of mAbs. they
are not the most suitable cells for large-scale processes of production,
since they lose their production ability over time.
• Production of urokinase- Urokinase is a serine protease that activates
plasminogen into plasmin, which in turn degrades fibrin clots. Hence
urokinase finds its value as an important anti-thromboembolic drug. The
need for urokinase production has increased significantly in recent years.
Mammalian cells provide post-translational modifications; therefore
mammalian cell lines are nowadays preferred for the production of
recombinant urokinase. CHO cells are suitable hosts for recombinant
urokinase production because they grow exceptionally well inside the
bioreactor.

12. • Production of follicle-stimulating hormone - Follicle-stimulating hormone
(FSH) is produced by the gonadotropic cells of the anterior pituitary. FSH
plays its role in follicular growth and development in females. It is essential
for males for the pubertal initiation and maintenance of quantitatively
normal adult spermatogenesis. Besides their therapeutic application in the
treatment of infertility, recombinant gonadotrophins have been used for
studying the structure and functions of glycoprotein hormones. Many groups
have prepared and described the actions of human recombinant (rec) FSH
produced by transfected CHO cells.
• Other applications of mammalian expression system - Novel mammalian cell
lines expressing reporter genes for the detection of environmental
chemicals activating endogenous aryl hydrocarbon receptors or estrogen
receptors were reported. Hemophilia B is a coagulation system genetic
disease affecting one in 30,000 males worldwide. Recombinant human Factor
IX (rhFIX) has been used for hemophilia B treatment. Stable and high-level
production of recombinant Factor IX in the human hepatic cell line was
reported

13. LIMITATIONS OF MAMMALIAN EXPRESSION
SYSTEM
• It is highly expensive.
• It is a complicated technology.
• Potential contamination with animal viruses of mammalian cell
expression has been a bottleneck for its use in large-scale industrial
production.
An optimal expression system can be selected only if the productivity,
bioactivity, purpose, and physicochemical characteristics of the interest
protein are taken into consideration, together with the cost,
convenience and safety of the system itself. It is the present need to
discover more mammalian cell lines and vector systems and technologies
to express the gene to get more efficient proteins having high biological
activities.

14. STRONG PROMOTERS
WHAT IS A PROMOTER?
A promoter is a DNA sequence where the RNA polymerase binds to initiate transcription. They are
present in both eukaryotes and prokaryotes.
• The strength of a promoter is the rate of transcription of the gene controlled by this promoter. The
strong or active promoter means the rate of transcription is high; and the weak or inactive promoter
means the rate of transcription is relatively low.
• A promoter is considered weak or strong based on the affinity for RNA polymerase and the sigma
factor. This means the consensus sequence of the promoter should match that of the RNA
polymerase. If it doesn’t exactly match, we can say that the promoter is weak.
• A weak promoter gives low transcription and thus low levels of the protein product. Now there
should be reasons why both weak and strong promoters are found in nature. It is because nature
doesn’t want certain genes to be active all the time.
• A strong promoter produces lots of gene products which results in a significant metabolic load. And
the cell has to find more resources and it’s difficult. This will not favor the organism in natural
selection and that's why weak promoters are obvious in nature.

15. PROMOTER REGION OF A GENE

16. • One way to know whether a promoter is strong or weak is by using a
reporter gene. Reporter genes when added give a quantifiable
phenotypic effect.
• An example of weak and strong promoters are promoter of lac operon
and promoter of lac repressor gene respectively. Lac operon is strong
since it needs to produce more b- galactosidase to break down lactose.
Now lac repressor gene makes sure repressor protein is made when
lactose is not present. This protein will inactivate the lac operon.
• Viral promoters are good. But the commonly used ones are plasmid
vectors. Viral promoters can be used almost everywhere. It is stable
under a wide range of organisms
• EXAMPLE- T7 RNA polymerase is very selective and efficient,
resulting both in a high frequency of transcription initiation and
effective elongation. These features result in an RNA elongation that is
approximately five-fold faster than for E. coli RNA polymerase; hence,
the T7 promoter is a much stronger promoter than the E.
coli promoters

17. REFERENCES
• REVIEW ARTICLE- Gene Expression in Mammalian Cells and
its Applications.
• https://www.thermofisher.com/in/en/home/life-science/protein-
biology
• https://www.creativebiomart.net/mammalian-expression-systems.htm
• https://www.creative-biolabs.com/drug-
discovery/therapeutics/mammalian-cell-expression-system.htm
• https://febs.onlinelibrary.wiley.com/doi/full/10.1111/j.1742-
4658.2010.07991.x
• https://www.researchgate.net