Assignment:

Subject Title:
Introduction To Biotechnology
BIT-405
Submitted To:
Dr. Aysha Yasmin
Submitted By:
Group (10)
Qamar Shehzad (6137)
Talha Shahzad (6123)
Haseeb Shokat (6111)
M Usman Shafiq (6153)
BS Biochemistry
8th (Morning)
Topic :
Recombinant Protein production and Purification

Government College University Faisalabad

 Recombinant Protein Production

1. Introduction:
Proteins are essential components of the living Things. They play a vital role as passive
structural elements as well as active functional units. Proteins constitute more than half of the
cells dry weight, comprise fundamental components of the cell structure and at the same time
serve as the main instruments of molecular recognition and catalysis. Although DNA serves as
the genetic template, it is the proteins it codes that influence almost all of the cellular processes.
For example, the genes encoding haemoglobin subunits cannot carry oxygen, the property of the
proteins specified by the genes . In nature proteins are synthesized via a sophisticated
mechanism of guided polymerization of simpler building blocks, the amino acids. The natural
protein production process is entwined with what is known as gene expression, the process in
which the information encoded in the hereditary polymer DNA is translated. In fact, the specific
polymerization order of a protein's amino acids has to be guided by the particular order of the
building blocks of a corresponding DNA. Proteins are expressed in the living organisms
according to a tightly regulated mechanism to fulfill specific needs and the process is described
as homologous gene expression. When recombinant DNA techniques are used to express certain
genes outside their natural expression habitat, the process is described as heterologous gene
expression. This process has various academic as well as industrial applications. Proteins may be
heterologous synthesized in vivo or in vitro. In vivo heterologous expression systems include any
nonnative cellular system that permits synthesis of the target protein, while the in vitro
heterologous expression systems include any, non-native, acellular system which permits such
synthesis. The study of different proteins and the increasing knowledge of their properties has led
to a growing number of protein products of commercial and medical use from domestic cleaning
to advanced bone and cartilage repair. Neither the fundamental study of a protein or its
development as a commercial product would have been possible without the availability of
sufficient protein in the first instance. (Mehmoud, 2007)

2. Basic steps to get recombinant Protein:

1. Amplification of gene of interest.
2. Insert into cloning vector.
3. Sub cloning into expression vector.
4. Transformation into protein expressing host (bacteria (E coli), yeast, mammalian cells or
baculovirus-insect cell system).
5. Test for identification of recombinant protein.( Western blot or Fluorescence)
6. Large scale production. (Large scale fermentation)
7. Isolation and purification. (Yilmaz H, 2019)
 3. Protein Expression Systems:

For the production of recombinant proteins, the gene is isolated and cloned into an expression
vector. Most recombinant proteins are from humans but are expressed in other organisms such as
bacteria, yeast, or animal cells in culture.
Many issues affect the expression of recombinant proteins because of organisms difference. The
gene for a recombinant protein can also be altered to make the protein more stable. Usage of
regulated expression vectors can control the rate at which recombinant protein is produced.
Another factor is protein export. It is easier to isolate and purify recombinant proteins if they are
exported outside the cell.
Many recombinant proteins require protein modifications, such as glycosylation, that are
available only in eukaryotic cells; this sometimes leads to the use of yeast, insect cells, and
mammalian cell culture systems.
A major concept in the production of recombinant proteins is the choice of expression
system(Host). The three major main systems for the expression of recombinant proteins are
bacterial expression systems (e.g. Lactococcus lactis and Escherichia coli), yeast and
baculovirus and mammalian cell mediated protein expression.

3.1. Bacterial Expression Systems:

What is Bacterial Protein Expression System?

The first choice of an expression system for the production of recombinant proteins for many
investigators is E. coli expression system. In other words, E. coli protein expression system is
now the most wide and common-used and economical expression system. Many proteins require
special modifications that are available only in eukaryotic cells. Disulfide bonds are not usually
formed inside bacterial cells and are rarely formed properly even when bacterial hosts secrete
eukaryotic proteins. In Figure3.1 Bacterial hosts are the most widely used expression system
due to the simple culture conditions and extremely rapid production of recombinant proteins
which is achieved through their use. This, combined with low production costs, leads to high
scalability, making these systems an attractive option for the production of recombinant protein.
 Fig (3.1) Bacterial expression system(Host)

3.1(a) Advantages of Bacterial Expression System.

The E. coli expression system has many advantages compared with other expression systems
such as easy growth conditions and rapid biomass accumulation. Expressing eukaryotic proteins
in eukaryotic cells that contamination with bacterial components can be avoided. Despite
purification, bacterial components that are toxic or promote immune reactions may be present in
traces if bacteria are used for production. Furthermore, some eukaryotic proteins are unstable or
inactive after being made by bacterial cells.
 3.2. Protein Expression in Mammalian Cells:
What is Mammalian Cells Expression System?
Some cloned animal genes may need to be expressed in cultured mammalian cells. Mammalian
shuttle vectors contain an origin of replication and an antibiotic resistance gene for growth in
bacteria. In other words, mammalian shuttle vectors have features for producing recombinant
proteins in mammalian cell cultures. Because most antibiotics do not harm mammalian cells, a
different method of selecting the mammalian shuttle vector must be used. In Figure 3.2
Transient and semi-stable mammalian antibody expression allows fast and parallelized
production without any need to generate producer cell lines. Transient antibody production is
suitable for small-scale production in antibody screening. The production volumes of transient
mammalian antibody production can be scaled up by employing batch or fed-batch bioreactor
processes to more than 150 L. The human embryonic kidney (HEK) 293 cell lines have been
widely used for transient protein expression. (Lai et al. 2013)

Fig (3.2) Mammalian cell expression systems (al., 2013)

3.2(a) Mammalian Cell Lines Yield High Levels of Recombinant Protein.

A major issue with protein expression in mammalian cells is finding a cell with high levels of the
recombinant protein. This single chosen cell is then used to generate large cultures. This ensures
that the recombinant protein is identical throughout the culture. Many techniques are available to
isolate single cells with high levels of recombinant protein such as limiting dilution cloning, cell
sorting, gel microdrop technology and automated cell selection.
From Figure 3.2 Our HostOptim™ technology has developed from our core expertise in the
field of gene expression and protein synthesis. Improved promoters, vectors and other
transcriptional approaches (i.e. more protein per mRNA) are used to increase expression levels.
This selection and expansion process takes about 2-3 months.

3.3. Protein Expression in Baculovirus Expression System:

What is Baculovirus Expression System?

The baculovirus-mediated insect protein expression system is based on the ability of recombinant
baculovirus to infect insect cells, and the foreign protein that is encoded by the baculovirus is
expressed by the cellular machinery. Insect cells are easy to grow for making recombinant
protein. Insect cells are infected with a virus genome that has the recombinant protein gene.
Rather than making viral particles, the infected insect cells make recombinant protein. (J, 2012).
In Figure 3.3 A flow chart of Baculovirus expression system presented. Briefly, a gene encoding
the protein-of-interest is inserted into a
Fig (3.3) Flowchart for recombinant protein expression in insect
cells.
primary vector, which is subsequently cloned into a secondary vector known as Bacmid. Bacmid
is transferred into a bacteria strain (commonly E.coli) for preliminary virus production and
assembly to obtain generation 1 baculovirus (P1). The P1 virus is amplified in an insect cell (e.g.,
sf9) to achieve a suitable titer (P2), which is then used to infect the same or a different insect cell
line (e.g., High-five) for protein expression. This “Bac-to-Bac” system has been adapted for the
expression of a wide variety of proteins as secreted, intracellular, or membrane-bound molecules.
Of note, there are many commercially available methods for baculovirus generation.
In Table 1 we are summarized commonly used of advantages and limitations of expressed hosts.
Proteins, the key products of the central dogma of molecular biology, are the basic building
blocks of life. Proteins perform various functions such a screating cellular matrices, catalyzing
biochemical reactions, and forming signaling pathways to respond to external stimuli. Studies of
the structure and function of proteins hold the key to understanding the “meaning of life,” so
they are pursued by scientists across multiple disciplines. However, because most proteins of
interest are difficult to obtain, it is important to establish sources that could potentially provide
researchers with unlimited supply of proteins. Recombinant protein expression is the process in
which a gene encoding a protein-of-interest is cloned into an expression vector (usually a
plasmid) and transferred into a host cell for protein production by harnessing its intrinsic protein
synthesis machinery.Several host cell systems have been established for recombinant protein
production and the selection of the optimal host for any given protein is a major factor for its
successful expression.

Table: Commonly used Host Cells for recommended protein expression.

(al, 2018)
 4. Protein Purification Methods:
Protein purification means protein fractionation which is also called downstream processing.
Protein purification involves a number of processes, including pumping and ultrafiltration, which
involve significant shear environments. More importantly, protein tags are a useful and
convenient tool for improving solubility of recombinant proteins, streamlining protein
purification, and allowing an easy way to track proteins during protein expression and
purification.
A wide variety of protein purification methods that can be combined to generate a suitable
purification scheme are available. Usually, one executes a series of purification steps, and only
rarely proteins can be purified in a single step.
Early steps combine low-resolution and high-capacity methods at later stages of purification
scheme. For low-resolution protein purification, methods such as fractional precipitation and
two-phase partition systems usually employed. For applications requiring the highest purity and
relatively small Protein Purification amounts of protein, chromatography can be chosen to
selectively purify the target protein. (Yilmaz H, 2019)

5. Recombinant Protein Purification:

What is Protein Purification?

The expression of recombinant proteins, especially using bacterial vectors and hosts, is a mature
technology. The problem is how to isolate it in an active form.
Purification of recombinant protein is an important technology in biological research. To study
the particular function and structure of a protein, researchers must isolate and purify the
recombinant protein from the organism. The protein purification method mainly uses the
similarity and difference between different recombinant proteins. Non-proteinaceous materials
can be removed based on the similarity between proteins, and the target recombinant protein then
can be isolated and purified based on the differences between proteins.
Protein tags are a banificial and convenient tool for improving solubility of recombinant proteins,
streamlining protein purification, and allowing an easy way to track proteins during protein
expression and purification. In addition, protein tags are a useful tool for tracking proteins and
processes directly in live cells using microscopy or indirectly using Western blot,
immunoprecipitation, or immunostaining. (PT, 2015)

6. Applications of recombinant proteins:

Recombinant proteins are proteins produced in an appropriate host system based on exogenous
DNA rather than the host's native DNA. The exogenous DNA, also called the gene of interest
(GOI), is introduced into the host cells through a suitable expression vector and then expressed to
proteins using the host genetic machinery.

Currently, recombinant protein production is one of the most powerful techniques used in life
sciences. Due to their advantages such as high purity, specificity, efficiency, safety,
customization, scalability, and consistency, recombinant proteins have been widely applied in
various fields, including medicine, research, biotechnology, etc.

1. Medicine.
2. Research.
3. Biotechnology.
4. Latest Research of Recombinant Proteins.
4.1.Recombinant Proteins and Diseases.
4.2.Recombinant Proteins and Vaccines.

1. Medicine:
Among protein-based biopharmaceuticals, recombinant proteins form the largest category,
encompassing enzymes, hormones, cytokines, growth factors, blood clotting factors, monoclonal
antibodies (mAbs), and antibody-related products (e.g., Fc-fusion proteins and antibody
fragments). They are used for therapeutics, diagnostics, and drug discovery and development, as
well as vaccine development and production.

1.1.Therapeutic Recombinant Proteins.

With the emergence of recombinant DNA technology, many therapeutic proteins that were
extracted from natural sources basically can be recombinantly produced in different expression
systems including bacterial, yeast, mammalian, and plant hosts.

Therapeutic recombinant proteins can be produced by modifying or altering the DNA via genetic
engineering techniques, providing important therapies for a variety of diseases, such as diabetes,
cancer, infectious diseases, autoimmune diseases, and genetic disorders.
1.1(a) Recombinant Enzymes.
In many genetic disorders, individuals may deficient functional enzymes due to genetic
mutations. Recombinant enzymes can be designed to mimic the missing or defective
enzymes. Recombinant glucocerebrosidase and recombinant alpha-galactosidase A are well-
established enzyme replacement therapies (ERT) for Gaucher's disease and Fabry'disease,
respectively.

1.1(b) Recombinant Cytokines and Growth Factors.

Some genetic disorders involve deficiencies in cytokines (encompassing molecules such
as interleukins, interferons, and colony-stimulating factors), which are signaling proteins
regulating immune responses and cell growth. Recombinant cytokines can be administered to
compensate for these deficiencies. Recombinant erythropoietin (EPO) can stimulate red blood
cell production and is used in anemia treatment.

Some recombinant cytokines such as immune checkpoint proteins also play a role in
immunotherapy by amplifying immune response against certain types of cancer. They function
to stimulate the immune system, evoke immune cells, and enhance anti-tumor reactions.

1.1(c) Recombinant Hormones.

Genetic conditions may lead to hormonal imbalances. Recombinant proteins can be engineered
to mimic hormones and restore normal physiological functions. Recombinant insulin is used to
control and treat diabetes. Recombinant human growth hormone (rhGH) is used in stimulating
growth in children with growth disorders.

1.1(d) Recombinant Clotting Factors.

Hemophilia, a genetic disorder, involves deficiencies in blood clotting factors. Recombinant
clotting factors, such as factor VIII and factor IX, are essential for blood clotting and is used in
hemophilia treatment.

1.1(e) Recombinant Antibodies.

Recombinant antibodies, especially monoclonal antibodies (mAbs), have revolutionized the
treatment of various cancers, autoimmune diseases, and infectious diseases. Rituximab is a CD20
monoclonal antibody that is widely used for lymphomas. Trastuzumab (Herceptin) and
adalimumab (Humira) are used in the treatment of breast cancer and autoimmune diseases (like
rheumatoid arthritis), respectively.

1.2. Diagnostic Agents.

In immunoassays, recombinant proteins are used as antigens to identify specific antibodies in
patient samples, facilitating the diagnosis of infectious diseases, autoimmune disorders, and
allergies. Additionally, these proteins also serve as calibrators and controls in diagnostic assays
to ensure consistency and accuracy in measuring analyte concentrations. This is critical for the
reliability of diagnostic results.

1.3. Precision Medicine.

Recombinant proteins enable the development of targeted therapies tailored to specific molecular
targets or pathways, improving treatment outcomes while reducing side effects. Recombinant
technology allows for the customization of therapeutic proteins to match the specific genetic
variations present in individual patients.

1.4. Vaccines.
Recombinant proteins are engineered to express specific antigens derived from pathogens such
as viruses or bacteria, which are highly immunogenic and capable of inducing a protective
immune response. These recombinant antigens such as surface proteins, subunits, or epitopes, are
key components in various vaccine platforms, including protein subunit vaccines, virus-like
particle (VLP) vaccines, and conjugate vaccines.

Recombinant protein vaccines not only show a high safety profile but also are more stable in
comparison to mRNA vaccines. Recombinant hepatitis B surface antigen (HBsAg) is used in
hepatitis B vaccines. Human papillomavirus (HPV) vaccines include recombinant virus-like
particles.

1.5. Drug Discovery and Development.

Recombinant proteins are used in high-throughput screening assays to identify potential drug
candidates. Researchers can efficiently test large compound libraries against specific targets,
expediting the drug discovery process.

Recombinant proteins are also used to design biological assays for studying the effects of
potential drug candidates. In preclinical studies, these recombinant proteins help researchers
understand the new drug's mechanism of action and assess its efficacy.

2. Research:

Recombinant proteins are essential tools in biological research, changing the way studying and
understanding cellular processes such as cell signaling, metabolism, growth, replication and
death, transcription, translation, and protein modification.

These proteins allow researchers to investigate the functions of specific genes and proteins in a
controlled environment. Recombinant proteins serve as valuable reagents in experiments, aiding
in protein purification, structural analysis, and investigation of protein-protein interactions,
receptor-ligand binding, and protein signaling pathways.
Recombinant proteins have proven performance in several laboratory techniques such as ELISA,
WB, and IHC. They can be used to develop enzymatic assays. When used in conjunction with a
matched antibody pair, recombinant proteins can be used as standards such as ELISA standards.
Moreover, recombinant proteins can be used as positive controls in WB.

3. Biotechnology:

Recombinant proteins are also used in industry, food production, agriculture, and biomaterials
and bioengineering.

3.1. Industry.
In the breeding industry, enzymes can be added to animal feed to increase the nutritional value of
feed ingredients, reduce feed and waste management costs, support animal gut health, enhance
animal performance, and improve the environment. Amylases are used in the food industry to
increase flavor, sweetness, and texture in various food items.

Cellulases are crucial enzymes in biofuel production, particularly in the conversion of

lignocellulosic biomass into biofuels such as bioethanol. Proteases play a crucial role in
detergent manufacturing, contributing to the effectiveness of laundry detergents and other
cleaning products.

3.2. Food Production.

Besides, lactic acid bacteria (LAB) have been used for a long time for the production of
fermented foods. LAB has recently been engineered for the expression of recombinant proteins,
which would have wide applications such as improving human/animal digestion and nutrition.

4. Agriculture.
Recombinant proteins have found various applications in agriculture, for example, crops can be
genetically engineered to express proteins that confer resistance to pests, diseases (caused by
viruses, bacteria, or fungi), or herbicides, contributing to improvements in crop yield and quality,
and overall agricultural sustainability.

5. Biomaterials and Bioengineering.

Recombinant proteins are used to design and produce functional biomaterials with tailored
properties such as bioactivity, biocompatibility, and structural integrity. By incorporating
specific protein sequences, these materials can mimic natural extracellular matrices, promoting
cell adhesion, proliferation, and tissue regeneration.

Recombinant proteins contribute to the field of tissue engineering by providing bioactive

components that support cell growth and tissue development. They can be engineered to mimic
the structural and signaling features of native tissues, facilitating the development of organ
transplantation.

5.1. Recombinant proteins also have limitations.

5.1(a) In some cases, the production of recombinant proteins is complex, expensive, and time-
consuming.

5.1(b) The recombinant proteins produced in cells may not be the same as the natural forms. This
difference may reduce the effectiveness of therapeutic recombinant proteins and even cause side
effects. Additionally, this difference may affect the results of experiments.

5.1(c) A major concern for all recombinant drugs is immunogenicity. All biotechnologically
produced therapeutics may exhibit some form of immunogenicity. It is difficult to predict the
safety of novel therapeutic proteins.
4. Latest Research of Recombinant Proteins:

There are several types of research on recombinant proteins from various fields that are describe
bellow.

4.1. Recombinant Proteins and Diseases.

4.1(a) Machado et al. designed a recombinant multiepitope protein for diagnosing

Chagas disease (rTC) and assessed the discrimination of clinical forms and cross-
reactivity . Their findings indicate that rTC can diagnose T. cruzi-infected individuals
with low cross-reactivity and high sensitivity and specificity, suggesting its potential as a
valuable component in the production of immunodiagnostic kits for Chagas disease.

4.1(b) Haiqiang Zhang et al. generated a HER-2-targeting recombinant protein that was
in tandem with HER-2-specific single-chain variable fragment (scFv), CCL19, and IL-7
(HCI fusion protein) can be stably obtained from transfected HEK-293 T cell strains.

In this study we confirmed that HCI fusion protein can facilitate the synergistic antitumor
effect of the immune system and that its increases antitumor ability produced
breakthrough effects on the tumor microenvironment (TME).

4.2. Recombinant Proteins and Vaccines.

4.2(a) Dan Liu et al. discovered that the immunogenicity of full-length recombinant
TprK (rTprK) against Treponema pallidum subsp. pallidum (T. pallidum) effectively
slowed lesion progression and decreased the T. pallidum burden .
 4.2(b) Immunization with rTprK not only promptly elicited a robust Th1-like cellular
response but also generated opsonic antibodies to increase macrophage-mediated
opsonophagocytosis. This study underscored the significance of TprK as a crucial
component in the development of a syphilis vaccine.

4.2(c) They also observed that the combination of rLF with inactivated Staphylococcus
aureus or recombinant FLIPr protein could increase mucosal and systemic immune
responses to inactivated virus or recombinant protein. These results suggest the potential
of rLF as an adjuvant and a vaccine candidate to block the immune evasion function of
FLIPr.

Overall, advancements in the field of biotechnology have increased and facilitated the production
of recombinant proteins for various applications. Although recombinant proteins still have some
drawbacks, their roles in medicine, research, and biotechnology are irreplaceable. We also look
forward to seeing more progress in the treatment of various diseases with recombinant proteins.
(Machado, , Juliana Martins, , & et al., 2023)

References
1. Mehmoud, K. (2007). Recombinant Protein Production:. Recombinant Protein Production Strategic
Technology and a Vital Research Tool.

2. al, L. e. (2018). Recombinant Protein Expression Using a Baculovirus-insect Cell System.

3. al., L. e. (2013). Mammalian Cell Expression System.
4. J, Z. (2012). Mammalian cell protein expression for biopharmaceutical production. Biotechnol Adv
30(5): 1158-1170.
5. Machado, , Juliana Martins, , & et al. (2023). Applications of recombinant proteins. Proof of Concept
of a Novel Multiepitope Recombinant Protein for the Serodiagnosis of Patients with Chagas Disease
[J].
6. PT, W. (2015). Overview of the purification of recombinant proteins. , Curr Protoc Protein Sci 80:
6.1.1-6.1.35.
7. Yilmaz H, e. a. (2019). Recombinant Protein Production. Production of recombinant n protein of
infectious bronchitis virus using the baculovirus expression system and its assessment as a diagnostic
antigen.