RECOMBINANT

RECOMBINANT
PROTEINS
PROTEINS
PRESENTED BY:
ASH . K
 INTRODUCTION
• Recombinant proteins are
the proteins that are
produced by genetically
modified organisms
following insertion of the
relevant DNA into their
genome.
• The science of
recombinant technology
took birth when Cohen &
Boyer (1973) were able to
introduce a piece of gene
containing foreign DNA
into plasmid of E.coli .

• TOOLS OF GENETIC
ENGINEERING :

ENZYMES

VEHICLE DNA

PASSENGER DNA
 RECOMBINANT DNA
TECHNOLOGY
• It involves following
steps :
1. Isolation of DNA
segment
2. Formation of
Recombinant DNA
3. Production of multiple
copies of recombinant
DNA
4. Introduction of
recombinant DNA into
host
5. Screening of the
transformed cells
 NEED & APPLICATION :
• Recombinant protein expression is the foundation
of today’s biomolecular research and the thriving
Biotech industry.
• GOAL: Overproduction of proteins for

Structural Enzymatic
studies studies

Antigen
Commercial/ production
Pharmaceutical
applications
• Genetic engineering produces proteins that
offer advantages over proteins isolated
from other biological sources.
• These advantages include:

• High Purity
• High Specific Activity
• Steady Supply
• Batch to Batch Consistency
Examples of Recombinant
Protein Products
1. HORMONES
• Insulin
• Human Growth Hormones
• Erythropoietin
•
2. BLOOD CLOTTING FACTORS
Coagulation factor VIII
Coagulation factor IX

3. IMMUNIZATION AGENTS
Hepatitis B vaccine

4. RESEARCH ENZYMES
Restriction endonucleases

5. INTERFERONS
 Protein Production-
Expression Systems
1. E.coli
2. Yeast
3. Filamentous
Fungi
4. Mammalian Cells
5. Plant cells
6. Insect cells
 E.coli
ADVANTAGES DISADVANTAGES

Fast growing missing or incorrect post-

translational modifications e.g.
missing glycosylation
Easy to perform •endotoxin contamination

Easy & cheap culture •Difficult purification (inclusion

bodies)

High yield producer

• Low yield of functional protein
 INSECT CELLS
Advantages Disadvantages
•easy to infect glycosylation not as complex as in
human systems

• stable integration (due to virus) •viral proteases degrade target

protein

easy purification: cells lyse by •optimization required to establish

themselves after 96 h (due to viral transfection
virus)
reliable protein folding

• high yields
 MAMMALIAN CELLS
ADVANTAGES DISADVANTAGES

• (Almost) human glycosylation and •so far - transfection may be

phosphorylation pattern difficult

•highest functionality production rates may be comparably

low
lowest immunogenicity and very high •selection of single clones is very
compatibility to humans time-consuming

if non-human cells - not susceptible higher costs for culture

for human pathogenes
• high safety profile, easy
permission as a drug
SUMMARY: Protein production – expression systems

Mammalian cell lines

Quality of protein
Handling & costs

Speed of process
Insect cell lines

Bacterial expression
systems

–
60-70% of all recombinant protein pharmaceuticals are
produced in mammalian cells
 WHY SYNTHESIZE HUMAN
INSULIN ?
•Protein hormone produced by beta cells of islets of Langerhans in
the pancreas

•Regulates blood sugar by allowing uptake of glucose from

bloodstream into body cells

•Patients with diabetes have insufficient or impaired production

of insulin
•Patients’ immune systems do not produce antibodies against human
insulin as they do with bovine or porcine insulin

•Projected decline in the production of animal-derived insulin

•Need for a more reliable and sustainable method of obtaining the

• product.
 STRUCTURE OF INSULIN

• Two polypeptide chains;

one with 21 amino acids
and the second with 30
amino acids

• Chains are linked via a

disulfide bond

• Gene encoding the

insulin protein is found
on chromosome 11
 Production of recombinant insulin
in E.coli
Human Insulin
(Humulin) is the
first therapeutic
product
produced by
recombinant
technology by Eli
Lilly & Co in
1980.
• STEPS
1. Synthesis of artificial genes coding for A & B chains of insulin
2. Construction of 2 recombinant plasmids
3. In each case artificial gene was ligated to a lacZ’ reading
frame present in a pBR322-type vector
4. The recombinant plasmids were separately transferred into
E.coli cells which secreted fused B-galactosidase-A chain &
B-galactosidase-Bchain separetely
5. These chains were isolated by detaching from B-galactosidase
in pure form
6. Detachment of proinsulin could be possible when an extra
methionine codon was added at the N’- terminus of each A & B
chains
7. The 2 chains A & B were joined invitro to reconstitute the
native insulin by sulphonating the 2 peptides with Na
disulphonate & Na sulphite.
8. Final product is Humulin - chemically identical to
human insulin
Synthesis of Human Growth
hormones in E.coli
• Somatostatin & Somatotropin are the 2 proteins
that act in conjugation to control growth
processes in human body, their malfunction leads
to painful and disabling disorders such as
acromegaly ( uncontrolled bom growth and
dwarfism)
• Early research showed that the condition could be
treated w/ injections of human growth hormone
 Growth hormone could only be obtained from
human pituitary glands
 These were obtained from cadavers
 Later studies showed that the cadaver supplied
GHs were often contaminated, so other methods
needed to be developed to artificially produce
human growth hormone
 Production of recombinant
Somatostatin
• Being a very short
protein, only 14 a.a in
length, it was ideally
suited for artificial
gene synthesis.

• Strategy involved
 Insertion of
artificial gene into
lacZ’ vector
 Synthesis of fusion
protein
 Cleavage with
cyanogen bromide
 Production of recombinant
Somatotropin
• Somatotropin presented a more
difficult problem
• This protein is 191 a.a in length,
equivalent to almost 600 bp, a
dificult prospect for today’s DNA
synthesis capabilities
• Infact a combination of artificial
gene synthesis and cDNA cloning
was used to obtain a somatotropin-
producing E.coli strain.
• mRNA was obtained from pituitary
gland & a Cdna library prepared
• Somatotropin Cdna turned out to
have a unique site for the
restriction endonuclese HaeIII,
which cuts the gene into 2
segments
• The longer segment, consisting of
codons 24-191, was retained for
use in construction of the
recombinant
plasmid
• The smaller segment was
replaced by an artificial
DNA molecule that
reproduced the start of
the somatotropin gene and
provided the correct
signals for translation in
E.coli

• The modified gene was

then ligated into an
expression vector carrying
the lac promoter
• Production of vaccines through recombinant DNA technology 1.INJECTABLE
HEPATITIS B VACCINE
• 2. EDIBLE VACCINE
 PROTEIN PURIFICATION

• Protein purification is a series of processes intended to

isolate a single type of protein from a complex mixture.

• Protein purification is vital for the characterization of

the function, structure and interactions of the protein of
interest.

• The various steps in the purification process separate

the protein and non-protein parts of the mixture, and
finally separate the desired protein from all other
proteins.

• Separation steps exploit differences in protein size,

physico-chemical properties and binding affinity
Methods of Protein Purification

 Precipitation and differential solubilization

 Ultracentrifugation

 Chromatographic Methods
 Affinity Chromatography
 Ion exchange chromatography
 HPLC
 AFFINITY CHROMATOGRAPHY

• Affinity chromatography
is a method of separating
biochemical mixtures,
based on a highly specific
biologic interaction such
as that b/w Ag & Ab,
enzyme & substrate or
receptor & ligand
 List of Recombinant Proteins
• Human Recombinants that largely replaced animal or
harvested from human types
 Human growth hormone(rhGH) Humatrope from Lilly & Serostim from
Serono replaced cadaver harvested hGH
 Human insulin(rhI) Humulin from Lilly & Novo Nordisk among others;
largely replaced bovine & porcine insulin for human therapy
 Factor V111 Kogenate from Bayer replaced blood harvested factor V111

• Human Recombinants with Recombination as only source

 Erythropoietin(EPO) Epogen from Amgen
 Granulocyte colony-stimulating factor ( G-CSF) sold as Neupogen from
Amgen
 DNAse by Genetech
 Tissue plasminogen activator (TPA) Activase by Genetech
• Animal Recombinants
 Bovine somatotropin (bST)
 Porcine somatotropin (pST)
 Bovine Chymosin

• Viral Recombinants
 Envelope protein of the hepatitisB virus marketed as Energix-B by
SmithKline Beecham