1.
COMMERCIALLY AVAILABLE RECOMBINANT PROTEINS
PRODUCTION OF INSULIN, GONADOTROPINS, GH, and HBsAg

2.
TOPICS COVERED THROUGH THIS PRESENTATION
Introduction to Recombinant Proteins
Insulin
Gonadotropins
Growth Hormones
HBsAg
Research Article with results
Conclusion and application
2

3.
What are recombinant proteins?
Recombinant proteins are proteins encoded by recombinant DNA that has been cloned in an
expression vector that supports expression of the gene and translation of messenger RNA.
Modification of the gene by recombinant DNA technology can lead to expression of a mutant protein.
Recombinant protein is a manipulated form of native protein, which is generated in various ways in
order to increase production of proteins, modify gene sequences, and manufacture useful commercial
products.
How are they made ?
Recombinant protein production begins at the
genetic level, where coding sequence for the
protein of interest is first isolated and cloned into
an expression plasmid vector. Most recombinant
proteins for therapeutic use are from humans but
are expressed in microorganisms such as bacteria,
yeast, or animal cells in culture. Human genes are
very complex, often containing non-coding DNA
sequences known as introns. Many recombinant
proteins require protein modifications, such as
glycosylation, that are available only in
eukaryotic cells. Yeast, insect cells, and
mammalian cell culture systems offer such post-
translation modifications.
Currently, most recombinant therapeutic proteins are
produced in mammalian cells because mammalian cells
are capable of producing high-quality proteins similar
to the naturally occurring ones. In addition, many
approved recombinant therapeutic proteins are
generated in E.coli due to its well-characterized
genetics, rapid growth, and high-yield production.
Tanya Singh

4.
What are recombinant proteins used for?
 Biomedical research to understand health and disease- Recombinant proteins are useful tools in understanding
protein-protein interactions. Protein interactions are fundamentally characterized as stable or transient and play
an important role in cellular processes. Recently, RP microarrays for examining protein-protein interactions have
become popular. For this approach, researchers seed a slide with numerous immobilized proteins, which they
then treat with a variety of molecules to examine how the two agents interact with one another. Using this
system, scientists have studied protein interactions with other proteins or peptides, enzymes, small molecules,
lipids, and nucleic acids. This allows for much higher throughputs when it comes to studying protein-protein
interactions.
 Recombinant proteins for bio-therapeutics- Most human diseases are systemically or partially related to
dysfunction of specific proteins. Therapeutic proteins provide important therapies for a variety of diseases, such
as diabetes, cancer, infectious diseases, hemophilia, and anemia. Common therapeutic proteins include
antibodies, Fc fusion proteins, hormones, interleukins, enzymes, and anticoagulants. Human proteins obtained
through genetic engineering play a key role in therapeutic medicines market. Recombinant proteins used in the
clinic include recombinant hormones, interferons, interleukins, growth factors, tumour necrosis factors, blood
clotting factors, thrombolytic drugs, and enzymes for treating major diseases such as diabetes, dwarfism,
myocardial infarction, congestive heart failure, cerebral apoplexy, multiple sclerosis, neutropenia,
thrombocytopenia, anaemia, hepatitis, rheumatoid arthritis, asthma, Crohn’s disease, and cancers therapies.
Tanya Singh 6

5.
RECOMBINANTINSULINPRODUCTION
INTRODUCTION
 Insulin has an essential role in glucose homeostasis. It is synthesised by
beta cells in the pancreas and has a fundamental role in fat and
carbohydrate metabolism.
 Human insulin is a 51 amino acid polypeptide. It contains two polypeptide
chains. The A chain has 21 amino acids and the B chain contains 30 amino
acids.
 Insulin has three disulfide bonds. Two of the disulfide bonds interlink the A
and B chains, whereas the third one is an intra A chain bond.
 The pro-hormone proinsulin, from which insulin is derived, contains 74
amino acids, Proinsulin secreted by the beta cells is relatively inactive
under biological conditions, but after cleavage in two places yields the two
chains (B and A) of the active hormone insulin, and the biologically inactive
C peptide.
 Porcine and bovine pancreatic tissue was the source of the hormone for
many years, followed by semisynthetic human insulin obtained by
modification of animal insulin.
 Human insulin was biosynthesized by Eli Lilly using recombinant DNA

6.
 Process development-The best growth conditions are identified to produce the most protein as
efficiently as possible.
 Process development starts with identifying cells that express the desired protein.
 It also includes the development and securing of resources, materials and tools for: the growth of
recombinant cells (upstream processing), isolation and purification of recombinant protein
(downstream processing).
 It also includes the selection of bioreactors, filtration equipment, liquid chromatography
equipment etc. This includes media, buffers, reagents and solutions and assays.
 At the end of process development, a working cell bank is laid and stored in liquid nitrogen.
 Manufacturing-. After successful cell line development and clone selection, small-scale cultures
using microtiter plates, test tubes, tissue culture flasks, and shake flasks are generally used for
screening of recombinant protein expression. It begins with defrosting a vial from working cell
bank and added in small amount of medium. Various cultivation parameters, such as media
composition, pH, agitation, aeration, temperature, cell density, the concentration of inducers,
induction time, and feeding strategies affect the protein expression level depending upon
expression
 After successful process development, large-scale production is carried out using bioreactor
systems to fulfill the demand for biopharmaceuticals. Batch, fed-batch, or continuous culture are
used for bulk production of recombinant proteins.
UPSTREAM PROCESSING

7.
Basic strategy of recombinant insulin production
Basic strategy of recombinant insulin production

8.
Methods of Insulin Production
Separate two chain method Proinsulin method
• The proinsulin biosynthesis strategy of human insulin is the preferred method. It is preferable to adopt
the “proinsulin route” as it only requires a single fermentation and purification procedure, which makes
the production process more efficient in comparison to the two-chain combination method

9.
Host and Vector for insulin production
1. E. coli is a preferred microorganism for large-scale production of recombinant proteins
 Advantages: high growth rate, simple media requirement, ease of handling, high yield, and
cost effectiveness
 Disadvantages:. Various post-translational modifications (PTMs) do not occur in E. coli and
Protein IBs formation. the inclusion body route of production from E. coli requires
complicated and extensive processing, such as solubilization and refolding procedures, to
obtain fully functional polypeptides.
 Vector: E.coli plasmid The recombinant gene of interest is usually carried on a high copy
number plasmid in order to boost gene dosage and, ultimately, process yield
 Advantages of bacterial plasmid: easy to handle, straightforward selection strategies, useful
for cloning small dna fragments
 disadvantage : cant uptake large dna molecules
2. Yeast
Advantages: can perform sufficient PTMs with their cell machinery, easy genetic manipulation,
cost-effective growth medium requirements.
Disadvantages: Compared to soluble expressions of bioactive therapeutic proteins, the inclusion
bodies have good mechanical stability and are resistant to proteolytic degradation.
Vector: yeast artificial chromosomes

10.
Applications
 Insulin is a hormone that is responsible for allowing glucose in the blood to enter cells,
providing them with the energy to function. It controls how cells and tissues absorb energy as
well as the breakdown of fats and proteins.
 A lack of effective insulin plays a key role in the development of diabetes.
 Insulin regular is used with a proper diet and exercise program to control high blood sugar in
people with diabetes. Controlling high blood sugar helps prevent kidney damage, blindness,
nerve problems, loss of limbs etc.
 non-diabetic uses of insulin include the following: wound healing, parenteral nutrition,
antiaging, body building, cardioprotection in acute coronary syndromes, insulin tolerance test
to test the hypothalamo-pituitary-adrenal axis functioning, cancer treatment, and
management of septic shock,

11.
Gonadotropin-releasing hormone
.It is also known as luteinizing hormone-releasing
hormone, a neurohormone consisting of 10 amino acids
that is produced in the arcuate nuclei of the hypothalamus.
.GnRH stimulates the synthesis and secretion of the two
gonadotropins—luteinizing hormone (LH) and follicle-
stimulating hormone (FSH)—by the anterior pituitary
gland.
.The effects of GnRH on the secretion of LH and FSH are
not exactly parallel, and the variations are probably due to
other modulating factors such as the serum concentrations
of steroid hormones.
. Characteristic of all releasing hormones and most
striking in the case of GnRH is the phenomenon of
pulsatile secretion.

12.
.Under normal circumstances, GnRH is released in pulses at intervals of about 90 to 120
minutes. In order to increase serum gonadotropin concentrations in patients with GnRH
deficiency, the releasing hormone must be administered in pulses.
.The neurons that secrete gonadotropin-releasing hormone have connections to an area of
the brain known as the limbic system, which is heavily involved in the control of emotions and
sexual activity.
.Hypogonadism, in which the functional activity of the gonads is decreased and sexual
development is inhibited can be caused by a congenital deficiency of GnRH. Patients with this
type of hypogonadism typically respond to pulsatile treatment with the hormone.
.This disorder is called Kallmann syndrome and is usually caused by a mutation in a gene that
directs the formation of the olfactory (sense of smell) system and the formation of parts of the
hypothalamus.

13.
Vector selection and applications.
.The advent of recombinant DNA technology
enabled the production of large amounts of
human gene products, such as recombinant
gonadotropins, for pharmacological applications.
.Recombinant gonadotropins are manufactured
by inserting the genes for the alpha and beta
subunits of FSH, LH and hCG into expression
vectors that are transferred into Chinese
hamster ovary cell lines. Recombinant FSH
(rFSH) has been proven to be safe and effective
in the treatment of infertility.

14.
.Recombinant HCG has the benefit of improved local tolerance compared with the
urinary one. Recombinant LH is indicated for use in women with profound LH and FSH
deficiencies, such as hypogonadotropic women.
APPLICATIONS:
>Treatment of anovulatory infertility.
>Treatment of females with blocked fallopian tube.
>Treatment of male subfertility or related conditions.
CLINICAL APPLICATIONS:
>Cell surface receptor of LH and hGC is found in a number of non-gonadal tissue.
>These hormones may possess other than reproductive functions and it is expressed by no of
tissues before birth,hinting at a potential developmental role.
>Receptor levels in non-gonadal tissues are generally much lower than in gonadal tissues.

15.
Additional recombinant hormones now approved:
>Three additional recombinant hormones have recently gained marketing approval:
.Thyroid-stimulating hormone-Structurally,thyroid-stimulating homone(TSH OR
thyrotrophin)is classified as a member of the gonadotrophin family,although functionallyit
targets the thyroid gland as opposed to the gonads.
.Parathyroid hormone-Human parathyroid hormone(hPTH)is an 84 amino acid polpeptide that functions as a
primary regulator of ca and phosphate metabolism in bones.
.Calcitonin-It is a polypetide hormone that plays a central role in regulating serum ionized calcium and inorganic
phosphate levels.

16.
Human Growth hormone :-(production,
vectors and applications)
 The gene for human growth hormone
(hGH) is isolated from human pituitary
gland
 Insertion of whole hGH gene into plasmid
vector and Cloning into E.coli results into
production of biologically inactive
hormone because bacteria can translate
the region of gene that are not translated
in human thereby producing a
prehormone containing an extra 26 amino
acids which might be difficult to remove.
 Hence the segment of gene that codes for
the first 24 amino acids of hormone is
constructed chemicallly from blocks of
nucleotide.

17.
Production hGH by
recombinant DNA technology:-
 From the known amino acids
sequences of hGH, gene for
first 24 amino acids are
constructed chemically.
 These genes are constructed
in three small fragments and
they are joined by T4 DNA
ligase to get whole gene for
first 24 amino acids.

18.
 Isolation of mRNA for hGH is isolated
from human pituitary gland tissue.
 Using reverse transcriptase enzymes
complimentary DNA (cDNA) is
synthesized from mRNA
 The cDNA obtained by reverse
transcriptase process, is the gene for
hGH
 The full gene is cut with restriction
endonuclease enzyme to remove first
24 gene.
 Joining of synthetic gene and cDNA ,
in this step synthetic gene(gene for
24 amino acids) and cDNA are joined
in order to obtain full gene with its
own initiation codon (AUG).
 T4 DNA ligase join these genes.

19.
Selection of suitable
vector and
recombination:-
 Expression vector
phGH407 derived from
plasmid vector PBR322 is
used as carrier vector.
 hgH gene is ligated into
restriction site just
downstream of
Lac.promoter/operator
region of the expression
vector.
Selection and
recombination into
suitable host cell:-
 E.coli used as suitable host
cell.
 The recombinant expression
vector is then transformed
into E.coli.
 The recombinant E.coli then
starts producing hGH.
 The recombinant E.coli are
isolated from culture and
mass production by
fermentation technology to
obtain hGH.

20.
Application of recombinant human
growth hormone (hGH):-
 Treatment of children suffering from growth
deficiency
 Treat the patient with Turner’s syndrome
and chronic renal insufficiency.
 To treat patient with renal carcinoma.
 Bovine somatropin hormone is used to
increase milk production in lactating cows
and also to increase body mass of cattles.

21.
HBsAg
Production of Recombinant Hepatitis B virus Antigen, Vectors involved and its application

22.
Introduction
 Hepatitis B surface antigen (HBsAg) is a surface antigen on the Hepatitis B
virus. If it is found, along with specific antibodies, it means the person has
a hepatitis B infection.
 Hepatitis B is a serious liver infection caused by the hepatitis B virus
(HBV). Having chronic hepatitis B increases your risk of developing liver
failure, liver cancer or cirrhosis—a condition that causes permanent
scarring of the liver.
Producing recombinant HBsAg vaccine -
 Immunization with hepatitis B vaccine is the most effective means of
preventing HBV infection and its consequences .This requires large amount
of HBsAg. Presently the recombinant antigen is produced in yeast and
mammalian cells (Chinese hamster ovary cells).
 Once the genome sequence is known, the gene coding for HBsAg is directly cloned
into a shuttle expression vector that replicates in both, E.coli for the genetic
manipulation steps, and in yeasts such as Saccharomyces cerevisiae for the
production of recombinant antigen.
 Transcription of the gene encoding HBsAg is driven from a strong yeast promoter
and is stopped by transcriptional terminator present in vector. The vector also has a
leucine biosynthesis marker for selection in yeasts and a tetracycline resistance
marker for selection in bacteria.

23.
Production of recombinant HBsAg from E.coli and
vectors involved
 For production of recombinant HBsAG, The E. coli strains DH5α is used for the cloning and expression experiments.
The Hepatitis B gene is cloned and maintained in pUC57 cloning vector. Bacterial expression vector pET-28a is
selected to express the gene encoding for HBsAg protein in E. coli.
 Two stop codons are included in the vector at the C- terminal cloning site with kanamycin resistance gene as
selection marker and poly linker sequence with multiple cloning sites . This vector replicates in E. coli through its
pBR322 origin of replication. Plasmid DNA then isolated and purified.
 Bacterial expression vector pET-28a is selected to express the gene encoding for HBsAg protein in E. coli. It
contains T7 promoter driven expression of recombinant proteins with the addition of a 19 amino acid N-terminal
fusion tag containing a His tag followed by a thrombin protease cleavage site. Two stop codons are included in the
vector at the C- terminal cloning site with kanamycin resistance (nptII) gene as selection marker and poly linker
sequence with multiple cloning sites . This vector replicates in E. coli through its pBR322 origin of replication.
 The purified HBsAg protein is then analyzed by SDS-PAGE and The protein samples are run on 12 percent acrylamide
gel along with standard protein marker. Further the expression of the HBsAg protein is then confirmed by western
blot.

24.
• Hepatitis B surface antigen consists of the S protein, M protein (S and preS2 proteins), and L protein (S,
preS1, and preS2 proteins). In hepatitis B surface antigen (HBsAg), the preS domain (preS1 + preS2) plays a
key role in the human hepatitis B virus (HBV) infection of hepatocytic cells by facilitating HBV attachment
and entry.
• Several diagnostic and therapeutic materials and systems based on preS have been developed, including
inhibitors of HBV infection, HBV vaccines, diagnostic tools for HBV infection, and hepatocyte-targeting
delivery systems for diagnostic and therapeutic molecules.

25.
Application of recombinant HBsAg
 HBsAg is the most important serum marker for diagnosing HBV infection. The presence of HBsAg indicates
infection and the person is infected as long as HBsAg or HBV DNA can be detected in the blood.
 Genotyping of HBV can serve to screen the possible sources of intrafamilial transmission, and how
phylogenetic analysis of the hypervariable region of the HBV genome complements HBV genotyping when the
infection source can not be ascertained by genotyping.
 Hepatitis B screening is an important tool for early detection and treatment. It can prevent serious illness,
such as cirrhosis and liver cancer, and hinder the spread of infection.
Clinical applications of Recombinant HBsAg
 Since its discovery by Blumberg in 1965, hepatitis B virus antigen (HBsAg) is used as the fingerprint of
hepatitis B infection. HBsAg level reflects the transcriptional activity of the cccDNA (covalently closed
circular DNA) which is a special DNA structure that arises during the propagation of some viruses in the
cell nucleus.
 It is an important marker that might not only indicate active hepatitis B infection, but may also predict
clinical and treatment outcomes.
 To detect HBsAg in serum and saliva samples, an enzyme immunoassay, ELISA, time-resolved
fluoroimmunoassay (TRFIA), or chemiluminescence immunoassay is commonly used.

26.
In this review, the authors report
on unpublished pivotal clinical
studies with recombinant
Insuman documenting the
efficacy and safety of
semisynthetic and recombinant
Insuman, and comparing
recombinant Insuman with
marketed formulations of
Humulin® (Eli Lilly,
Indianapolis, Indiana, US).
Furthermore, the use of insulin
formulations in clinical practice
is discussed to review the place
of recombinant human insulin in
routine clinical practice of today.

27.
Comparison of Pharmacokinetic (A) and
Pharmacodynamic (B) Profiles of Semisynthetic
Regular Insulin and Recombinant Regular
Insulin (Insuman Rapid) after a Single Dose
Injection of 0.3 U/kg in 12 Healthy Subjects
Using the Euglycaemic Clamp Technique
• Recombinant Insuman was approved in Europe in
1997 and launched in 1999. The early clinical studies
are summarised in the EMA approval documents.
• The formulations comprise regular soluble insulin
(Insuman Rapid), an intermediate-acting insulin
suspension that contains isophane insulin (Insuman
Basal), and premixed combinations of regular insulin
and intermediate-acting insulins (Insuman Comb).
• During early studies, the bioequivalence of
formulations based on semisynthetic insulin and
recombinant Insuman was established subsequently
bioequivalence studies for comparison with marketed
recombinant insulin formulations were performed and
followed by the clinical studies reviewed here.

28.
Comparison of Serum Insulin Concentrations after Subcutaneous Singledose Injection (0.3 U/kg) of Two Insuman
Formulations in 24 Healthy Subjects from Two Clamp Studies.
sc- subcutaneous
In clinical emergency, Insuman Rapid may be injected or
infused intravenously under strict glucose control. When
using continuous subcutaneous insulin infusion (insulin
pumps), there is a clear clinical preference for fast-acting
insulin analogues in the paediatric population. Studies with
a specific formulation (Insuman Infusat) in external
portable insulin pumps have shown improved stability to
minimise loss of efficacy, which may result from
mechanical and thermal stress. A limited number of studies
have been performed for the use of Insuman Infusat in
external insulin pumps, and with Insuman Implantable in
intraperitoneal insulin pumps.45,46

29.
As the burden of diabetes is predicted to increase dramatically in the next decades, the demand of insulin in sufficient
amounts and at affordable costs remains a challenge, particularly in developing countries. For that reason, recombinant
human insulin formulations are considered to be effective alternatives to insulin analogues in countries with limited
healthcare systems and limited resources. Continuing patient education support programmes on disease management and
correct use of human insulins are essential (e.g. re-suspension of NPH and premixed formulations, injection technique,
adequate storage) for a key role of recombinant human insulins in diabetes treatment. In developing countries, improving
access to human insulin can support initiatives for earlier insulin therapy, to improve glycaemic control in the
communities, and finally to delay or reduce diabetes complications in the long term.

30.
Applications of recombinant proteins
1. Medicine
2. Research
3. Biotechnology
4. The Latest Researches of Recombinant Proteins
• Recombinant proteins and diseases
1. Medicine
Most human diseases are systemically or partially related to dysfuction of specific proteins. Therapeutic
proteins provide important therapies for a variety of diseases, such as diabetes, cancer, infectious
diseases, hemophilia, and anemia. Common therapeutic proteins include antibodies, FC fusion proteins,
hormones, interleukins, enzymes, and anticoagulants.
-Shafaq

31.
 Currently, most of all recombinant therapeutic proteins are produced in mammalian
cells because mammalian cells are capable of producing high-quality proteins similar
to the naturally occurring ones. In addition, many approved recombinant therapeutic
proteins are generated in E.coli due to its well-characterized genetics, rapid growth,
and high-yield production.
2. Research
Recombinant proteins are useful tools in understanding protein-protein interactions.
They have proven performance in several laboratory techniques, such as ELISA, Western
Blot, and immunohistochemistry (IHC). Recombinant proteins 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 Western blots.
3. Biotechnology
Recombinant proteins are also used in industry, food production, agriculture, and
bioengineering. For example, in 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.

32.
 Besides, lactic acid bacteria (LAB) have been used for a long time for the production of
fermented foods, and recently, LAB has been engineered for the expression of
recombinant proteins, which would have wide applications such as improving
human/animal digestion and nutrition.
4. The Latest Researches of Recombinant Proteins
● Alpha-1-antitrypsin, a protein made by the liver, is secreted to the bloodstream, and then
circulates the body to protect the lung. The patients who can not produce the protein
usually regularly and quantitatively receieve an infusion of Alpha-1-antitrypsin protein,
which is extracted from donor blood. Researchers from The Novo Nordisk Foundation have
successfully produced Alpha-1-antitrypsin in CHO cells at scale. Furthermore, the
researchers also improved the proteins by genetic techniques, thereby obtaining
recombinant therapeutic proteins similar to human versions with a specific sugar-structure.
It is likely to eliminate the need for human donors in the future.

33.
conclusion
 Recombinant protein expression and purification is frequently useful for both basic research and
commercial application . High-through put Protin expression and purification has begun to revolutionize
the manner in which studies are conducted in various research fields.
 Recombinant proteins are commonly used to produce pharmaceutical products, protein-based polymers
for drug delivery, antibodies and enzymes for disease treatment, protein scaffolds for tissue
engineering, as well as for a myriad of other uses.