1. EUKARYOTIC EXPRESSION
SYSTEMS AND THEIR
APPLICATIONS
Presented by-:
Vimlesh Gupta
L-2011-V-59-M
Department of Veterinary
Microbiology…

2. INTRODUCTION
 Expression system-:
 Overproduction of proteins by placing the gene
encoding them under the control of a strong
promoter.
 Goal:require for those proteins that are produced at
low level , for structural & functional studies and for
medical & industrial purpose.

3. THESE TECHNIQUES RELY ON:
 the universality of the Genetic Code
 knowing the Genetic Code
 the relative similarity of the translational
machinery (ribosome).
 Rapid progress in molecular
biology/genetic engineering
cloning/amplification .
 DNA sequencing.
 Cutting and pasting pieces of DNA from
one source into another.
• The Expression vector

4. EXPRESSION VECTORS
• It is engineered plasmid to contain regulatory
sequences that act as enhancer and promoter
regions and lead to efficient transcription of the
gene carried on the expression vector.
• Goal -is the production of large amounts of
stable messenger RNA, and therefore proteins.
 Origin of replication: DNA polymerase
 selectable marker(s): antibiotic resistance
 promoter: recognized by RNA polymerase
 multiple cloning sites (restriction enzyme
sites): cutting/pasting of DNA fragments.
 Termination site: to stop the transcription.

5. EXPRESSION OF TARGET GENE…..
 Once the expression vector
inside the cell, expression of
target genes occurs.
5‟ UTR 3‟ UTR
Protein Coding Region
G ExonExon 2 3
1 Exon AA
AA
5’ Cap A 3’
Poly
A Tail

6. PROTEIN EXPRESSION SYSTEMS

7. CHOICE OF THE EXPRESSION SYSTEM
Cell-free Bacteria Yeast Insect Mammalian
Easy of use
Cost of media and Equipment
Pos-translational Modifications
(Probability of protein function)
Time Requirement
7

8. EXPRESSION IN E. COLI
 E. coli remains the host cell of choice for the majority of
protein expression experiments.
 Its rapid doubling time (approximately 30 min) in simple
defined(inexpensive)media.
 Extensive knowledge of its promoter and terminator
sequences.
 Of course, E. coli does suffer from the fact that is a
prokaryotic organism when it is used to produce
eukaryotic proteins.

9. 1) Problems resulting from
the sequence of the
foreign gene
(a) Introns are not removed in E. coli
(b) Premature termination of transcription
(c) a problem with codon bias.

10. 2)PROBLEMS CAUSED BY E. COLI
 Unfortunately, the proteins of
bacteria and higher organisms
are not processed identically.
 E.g. Glycosylation of
recombinant proteins
synthesized in E. coli are never
glycosylated correctly.
 E. coli might not fold the
recombinant protein correctly, so
usually it is insoluble and forms
an inclusion body within the
bacterium.
 E. coli might degrade the
recombinant protein.

11. EXPRESSION IN EUKARYOTIC SYSTEM
 The problems associated with obtaining high yields
of active recombinant proteins from genes cloned in
E. coli have led to the development of expression
systems for other organisms.
 Yeast and filamentous fungi.
 Insects.
 Higher eukaryotic cells.

12. Four promoters frequently used in expression vectors for microbial
eukaryotes. P = promoter.

13. EXPRESSION IN YEAST
 As eukaryotes, yeasts have many of the
advantages of higher-eukaryotic cells, such as
post-translational modifications.
 Yeast cell growth is faster, easier and less
expensive than other eukaryotic cells, and generally
gives higher expression levels.
 Three main species of yeast are used for the
production of recombinant proteins –
Saccharomyces cerevisiae, Pichia pastoris and
Schizosaccharomyces pombe.

14. SACCHAROMYCES CEREVISIAE
 Baker‟s yeast, S. cerevisiae, is a single-celled
eukaryote that grows rapidly (a doubling time of
approximately 90 min) in simple, defined media
similar to those used for E. coli cell growth.
 Proteins produced in S. cerevisiae contain many,
but not all, of the post-translation modifications
found in higher eukaryotic cells.

15.  Saccharomyces cerevisiae is currently the most popular
microbial eukaryote for recombinant protein production.
 A number of strong constitutive promoters have been
used to drive target gene expression in yeast.
 For example, the promoters for the genes encoding
phosphoglycerate kinase (PGK), glyceraldehyde-3-
phosphate dehydrogenase (GPD) and alcohol
dehydrogenase (ADH1) have all been used to produce
target protein.
 The GAL promoter is induced by galactose,regulating
expression of a cloned foreign gene.

16. PROMOTERS FOR S. CEREVISIAE EXPRESSION
VECTORS

17. 1) THE GAL SYSTEM
 In yeast, like almost all other cells, galactose is
converted to glucose-6-phosphate by the enzymes
of the Leloir pathway.
 Each of the Leloir pathway structural genes
(collectively called the GAL genes) are expressed
at a high level, but only when the cells are grown on
galactose as the sole carbon source.
 Each of the GAL genes contains within its
promoter at least one, and often multiple, binding
sites for the transcriptional activator Gal4p.
 The binding of Gal4p to these sites, and its
transcriptional activity when bound, is regulated by
the source of carbon available to the cell.

18. When yeast is grown on glucose-:
 transcription from the GAL4 promoter is down-regulated
 Reduced production of Gal4p activator in the cell,
 reduced level of activator binding at the promoters of the GAL
structural genes.
Carbon sources, such as raffinose-:
 Gal4p is produced and binds to the GAL structure gene
promoters, but a repressor, Gal80p, inhibits its activity.
 Gal80p binds directly to Gal4p and is thought to mask its
activation domain such that it is unable to recruit the
transcriptional machinery to the gene.
Only in the presence of galactose-: is the inhibitory effect of
Gal80p alleviated, leading to strong, inducible levels of target
gene expression.

19.  To produce a target protein in S. cerevisiae using
galactose induction, the gene encoding the protein must
be cloned so that it is under the control of a GAL
promoter.
 The promoter from the GAL1 gene, encoding
galactokinase, is most commonly used, but synthetic
promoters containing multiple Gal4p binding sites are
also available.
 Once constructed, the expression vector is transformed
into yeast cells and protein production is initiated by
switching the cells into a galactose-containing medium.
Proteins produced in this way seldom accumulate to the
levels of recombinant protein found in E. coli cells.

20.  A difficulty is brought about as a consequence of the
activator of the GAL genes, Gal4p, being normally
present in the yeast cell at a very low level.
 Therefore, if the expression vector, which carries multiple
Gal4p binding sites, is a high-copy-number plasmid then
there may be insufficient Gal4p to activate the expression
of all of the available target genes to a maximum level.
 To overcome this problem-: GAL4, is also placed under
the control of PGAL1

21. Figure: Galactose inducible gene expression in yeast. The expression
of genes from multicopy vectors under the control of the GAL1 promoter
(PGAL1) can be increased substantially if the gene encoding the
transcriptional activator of GAL1, GAL4, is also placed under the control of
PGAL1. In this case, induction by galactose will produce more Gal4p and
consequently more of the target protein.

22. 2) THE CUP1 SYSTEM
 Cells must maintain a proper cellular level of copper
ions (Cu2+ and Cu+) that is not too low to cause
deficiency and not too high to cause toxicity.
 In S. cerevisiae, copper homeostasis consists of
uptake, distribution and detoxification mechanisms.
 At high concentrations, copper ion detoxification is
mediated by a copper ion sensing metalloregulatory
transcription factor called Ace1p.
 Upon interaction with copper, Ace1p binds DNA
upstream of the CUP1 gene, which encodes a
metallothionein protein, and induces its transcription.

23.  The transcription of CUP1 is induced rapidly by addition
of exogenous copper to the medium.
 Expression vectors harbouring the CUP1 promoter can
therefore be used to induce target gene expression in a
copper-dependent fashion.
 Advantage-:
 Unlike the GAL system, yeast cultures containing the
CUP1 expression plasmid can be grown on rich carbon
sources, such as glucose, to high cell density, and
protein production is initiated by the addition of copper
sulphate to the cultures.

24.  Disadvantage-:
 One potential drawback with this system is the presence
of copper ions in yeast growth media, and indeed in
water supplies.
 Therefore, the „off‟ state in the absence of added copper
may still yield significant levels of protein production
Yields of recombinant protein are relatively high.

25. Recombinant
Proteins
Successfully
Produced in S.
cerevisiae
•For a range of reasons
as expressed previously
each of these
represented a better
product than was
obtainable using a
prokaryotic expression
system

26. WHY OTHER YEAST SPECIES?
 S. cerevisiae sometimes hyperglycosylates proteins
-Proteins also sometimes retained in periplasmic space
 S. cerevisiae also produces ethanol at high cell
densities which is toxic to cells.

27. PichiaPastoris
 Highly efficient promoters available like-:
 AOX1 (alcohol oxidase for methanol metabolism) promoter
easily turned on by methanol
 The promoter regulating the production of alcohol oxidase
(AOX1) can be used to drive heterologous protein expression
in P. pastoris since it is tightly regulated and induced by
methanol to very high levels.
 The first step in the metabolism of methanol is the oxidation of
methanol to formaldehyde using molecular oxygen (O2) by the
enzyme alcohol oxidase.
 Alcohol oxidase has a poor affinity for O2, and P. pastoris
compensates for this deficiency by generating large amounts
of the enzymes.

28. P. PASTORIS INTEGRATING EXPRESSION
VECTOR
•Integrating system
requires double
recombination
(AOX1p and 3‟AOX1
regions)
•His selection in a
HIS4- strain
•Usual
prokaryotic/eukaryoti
c sequences
•Alcohol oxidase
expression system
(AOX1)

29.  Advantages-:
 High level of target protein- For example, the expression of the
gene encoding recombinant hepatitis B surface antigen results
in the production of more than 1 g of the antigen from 1 L of P.
pastoris cells. This is much greater than could be achieved in
S. cerevisiae.
 Additionally, in comparison to S. cerevisiae, P. pastoris may
have an advantage in the glycosylation of secreted proteins.
 Glycoproteins generated in P. pastoris more closely resemble
the glycoprotein structure of those found in higher eukaryotes.
 Low level of ethanol.
 Disadvantages-:
 The only significant problem with P. pastoris is that it
sometimes degrades recombinant proteins before they can be
purified.

30. SCHIZOSACCHAROMYCES POMBE
 S. pombe is a single-cell eukaryotic organism with
many properties similar to those found in higher-
eukaryotic organisms.
 Additionally, eukaryotic proteins expressed in S.
pombe are more likely to be folded properly, which
may reduce protein insolubility associated with the
production of many proteins in E. coli.

31.  Protein production in S. pombe is usually controlled by
the expression from the nmt1 (no message in thiamine)
promoter.
 This promoter is active when the cells are grown in the
absence of thiamine, allowing downstream transcription
of genes under its control, while in the presence of
greater than 0.5 μM thiamine, the promoter is turned off.
 Overall protein production levels are similar to those
found in S. cerevisiae.

32. EXPRESSION IN INSECT CELLS
 The expression system is based on the
baculoviruses, a group of viruses that are
common in insects but do not normally infect
vertebrates.
 The baculovirus genome includes the polyhedrin
gene, whose product accumulates in the insect cell
as large nuclear inclusion bodies toward the end of
the infection cycle .
 Similar levels of protein production also occur if the
normal gene is replaced by a foreign one.

33. Baculovirus Systems
 Baculoviruses are rod-shaped viruses that
infect insects and insect cell lines.
 They have double-stranded circular DNA
genomes in the range of 90–180 kbp.
 Viral infection results in cell lysis, usually
3–5 d after the initial infection, and the
subsequent death of the infected insect.
 The nuclear polyhedrosis viruses are a
class of baculoviruses that produce
occlusion bodies in the nucleus of infect
cells.
 These occlusion bodies consist primarily of
a single protein, polyhedrin, which
surrounds the viral particles and protects
them from harsh environments.

34. CONT..
•Polyhedron gene
is not essential for
life cycle (protects
virus in
environment)
•Commonly used
with cultured
insect eggs

35. BACULOVIRUS TRANSFER VECTOR
•Done in cell
culture

36. The production of a recombinant
baculoviral genome for the
production
of proteins in insect cells. The target
gene is cloned under the control of the
polyhedrin
promoter into a transfer vector that also
contains regions of the viral genome
that flank the
polyhedrin locus. The vector is then co-
transfected into insect cells with a viral
genome that
has been linearized using restriction
enzymes (RE) that cut in several places.
Homologous
recombination between the linear
genome and the vector will result in
formation of a
functional viral genome that is capable
of producing viral particles. The
inclusion of lacZ
in the transfer vector allows for visual
screening of viral plaques to identify
recombinants. recombinant virus look
different because they lack the coat
protein.

37. Baculovirus Expression System
.
.
usually contain the lacZ
gene, or another readily
observable reporter
gene, which allows for the
visual identification of
recombinant plaques by
their blue appearance after
staining with X-Gal.

38. ADVANTAGES
 The polyhedrin gene is not required for the continuous
production of infectious virus in insect cell culture. Its
sequence is replaced with that of the heterologous gene.
 The polyhedrin gene promoter is very strong. This determines
a very high level of production of recombinant protein.
 This system is capable of post-translational modifications.
Disadvantages
• Expensive.
• Glycosylation in insect cells is different.
• Discontinuous expression: baculovirus infection of insect cells kills
the host and hence the need to reinfect fresh cultures for each
round of protein synthesis.
• Inefficient for production on a commercial scale

39. EXPRESSION IN HIGHER-EUKARYOTIC CELLS
 For the production of mammalian
proteins, mammalian cells have an obvious
advantage.
 In most cases these proteins have been processed
correctly and are indistinguishable from the non-
recombinant versions.
• Two modes of expression - transient and stable.
• Three cell types are dominant in transient
expression: human embryonic kidney (HEK), COS
and baby hamster kidney (BHK), whilst CHO
(Chinese hamster ovary) cells are used
predominantly for stable expression.

40. MAMMALIAN EXPRESSION VECTORS
 Eukaryotic origin of replication -is from an
animal virus: e.g. Simian virus 40 (SV40).
 Selective marker-antibiotic resistance genes.
 Promoter sequences that drive expression of
both marker and cloned heterologous gene.
 The transcription termination
 Polyadenilation signals-are usually from
animal viruses (human CMV, SV40, herpes
simplex virus) or mammalian genes (bovine
growth hormone, thymidine kinase).

41. MAMMALIAN EXPRESSION VECTOR
•“I” is an intron that enhances expression
•Other signals similar to insect and prokaryotic
vectors

42. TWO VECTOR EXPRESSION SYSTEM
•Useful for proteins of two different polypeptides

43. TWO GENE EXPRESSION VECTOR

44. SELECTIVE MARKER GENE SYSTEMS FOR
MAMMALIAN CELLS

45.  These systems, however suffer from leaky gene
expression in the absence of induction and potentially
damaging induction conditions.
 To overcome some of the problems of using endogenous
promoters to drive target gene expression, systems have
been imported from bacteria to control gene expression
in mammalian cells.

46. TET-ON/TET-OFF SYSTEM
 The E. coli tet operon was originally identified as a
transposon (Tn10) that confers resistance to the
antibiotic tetracyclin.
 The TetR protein, in a similar fashion to the lac
repressor protein (LacI), binds to the operator of the
tetracycline-resistance operon and prevents RNA
polymerase from initiating transcription.

47.  Regulator plasmid – produces a version of the E. coli
tetracycline repressor (TetR) that is fused to the
transcriptional activation domain of the herpes simplex
virus VP16 protein. The fusion protein is constitutively
produced in the host cell from the CMV promoter.
 Response plasmid – contains the target gene cloned
downstream of multimerised copies of the tetracycline
operator (tetO) DNA sequence that form a tetracycline
response element (TRE) cloned into a minimal CMV
promoter that is not, on its own, able to support gene
activation.

48. Figure :Tetracycline
regulated gene
expression for protein
production in
mammalian cells.
The Tet-off and Tet-on
systems differ in their
transcriptional response
to added tetracycline.
The Tet-off system turns
transcription of the
target gene off in
response to
tetracycline, whereas
the Tet-on system, which
contains a mutant
version of TetR with
altered DNA binding
properties, activates
gene expression in
response to tetracycline

49.  In the absence of tetracycline, the TetR-VP16 fusion
protein will bind to the TRE and activate transcription of
the target gene.
 Upon the addition of tetracycline to the
cells, however, TetR will dissociate and target gene
transcription will be turned off.
 That is, the addition of tetracycline turns target gene
expression off.
 The use of the tet system has become more prevalent
due to the existence of a mutant version of TetR.

50.  Tet-off uses the wild-type TetR protein fused to VP16.
Target gene expression is active in the absence of
tetracycline but not in its presence.
 Tet-on uses the mutant rTetR proteins fused to VP16.
Target gene expression is active in the presence of
tetracycline but not in its absence.

51.  Advantages:
 The advantage of this on and off switching system is that host
cells do not need to be exposed for long times to the antibiotic
prior to the induction of either gene expression or gene
silencing.
 There are no examples of higher eukaryotic proteins, which
could not be made in detectable levels, and in a form identical
to the natural host (that includes all types of post-translational
modifications).
 Disadvantages:
 Cultures characterised by lower cell densities and lower
growth rates.
 Maintenance and growing very expensive.
 Gene manipulations are very difficult.
 Mammalian cells might contain oncogenes or viral DNA, so
recombinant protein products must be tested more
extensively

52. PHARMING—RECOMBINANT PROTEIN FROM LIVE
ANIMALS AND PLANTS
 The use of silkworms for recombinant protein
production is an example of the process Often
referred to as pharming, where a transgenic
organism acts as the host for protein synthesis.
 Pharming is a recent and controversial innovation in
gene cloning.

53. APPLICATIONS OF RECOMBINANT PROTEINS
 Eukaryotic expression systems are frequently
employed for the production of recombinant
proteins for structural & functional studies and for
medical & industrial applications like-:
 Hormones
 Insulin: Diabetes
 Human thyroid stimulating hormone
 Blood clotting factors
 Coagulation factor VIII : hemophilia A.
 Coagulation factor IX: hemophilia B.

54. CONT….
 Interferons
 interferon-(alpha)-2a: chronic hepatitis C.
 gamma interferon: hepatitis B, C, herpes and viral
enteritis.
 Immunization agents
 Hepatitis B vaccine: a non-infectious vaccine derived
from Hepatitis B surface antigen (HBSA) produced in
yeast cells.
 Research enzymes
 Restriction endonucleases
 Endoglycosidases: PNGase

55. Recombinant
Proteins
Successfully
Produced in S.
cerevisiae
•For a range of reasons
as expressed previously
each of these
represented a better
product than was
obtainable using a
prokaryotic expression
system

56. EXAMPLES OF PROTEINS SUCCESSFULLY
PRODUCED BY BACULOVIRUS SYSTEMS

57. Thank u..!