this is a presentation on gene expression vector that includes what is expression vector, how many types of expression vector and difference between cloning and expression vector

1. TOPIC- GENE EXPRESSION VECTOR
PRESENTED BY:
TAHURA MARIYAM
MSc. MICROBIOLOGY
PRENENTED TO: Dr. Gurudayal Ram (Assistant prof. Sr. Grade)
DEPARTMENT OF INDUSTRIAL MICROBIOLOGY
JACOB INSTITUTE OF BIOTECHNOLOGY AND BIO-ENGINEERING
SAM HIGGINBOTTOM UNIVERSITY OF AGRICULTURE,TECHNOLOGY, AND SCIENCES,
PRAYAGRAJ

2. Content
1. INTRODUCTION
2. ELEMENTS OF EXPRESSION VECTOR
3. ELEMENTS OF EXPRESSION
4. PROTEIN TAGS
5. OTHERS
6. EXPRESSION/PRODUCTION VECTOR
 BACTERIA
 YEAST
 CELL-FREE SYSTEM
7. APPLICATION
 PRODUCTION OF PEPTIDE AND PROTEIN PHARMACEUTICALS

3. Introduction
 An expression vector, otherwise known as an expression construct, is usually a plasmid or
virus designed for gene expression in cells. The vector is used to introduce a specific gene
into a target cell, and can commandeer the cell's mechanism for protein synthesis to
produce the protein encoded by the gene. Expression vectors are the basic tools in
biotechnology for the production of proteins.
 The vector is engineered to contain regulatory sequences that act as enhancer and
promoter regions and lead to efficient transcription of the gene carried on the expression
vector. The goal of a well-designed expression vector is the efficient production of protein,
and this may be achieved by the production of significant amount of stable messenger
RNA, which can then be translated into protein. The expression of a protein may be tightly
controlled, and the protein is only produced in significant quantity when necessary
through the use of an inducer, in some systems however the protein may be expressed
constitutively. Escherichia coli is commonly used as the host for protein production, but
other cell types may also be used. An example of the use of expression vector is the
production of insulin, which is used for medical treatments of diabetes.

4. Expression Vector
 The cloning vector containing suitable expression signals to have
maximum gene for expression is called expression vector.
 The following expression signals are introduced into gene cloned vectors
are get maximum expression
 Insertion of strong promoter.
 Insertion of strong termination codon.
 Adjustment of distance between promoter and cloned gene.
 Insertion of transcription termination sequence.

5. Choice of expression vector
 Strong promoter
 Intact ORF
 Ribosomal binding site
 Termination sequence

6. Expression in Prokaryotes
 Promoter-commonly used inducible promoter are promoters derived
from lac operon and the T7 promoter. Other strong promoters used
include Trp
 promoter and Tac promoter, which a hybrid of both the Trp and Lac
operon promoters
 Ribosome binding site(RBS) following the promoter, and promoter
efficient translation of the protein of interest.
 Translation initiation site-shine dalgarno sequence enclosed in the RBS,8
base-pair upstream of the AUG start codon.

7. Post-Translational Modification
 Eukaryotic protein synthesis occurs in cytoplasm or on the endoplasmic
reticulum.
 These protein are further are further post translation processed that is
required for protein activity and stability.
 Disulfide isomerase also make sure that the proteins produced have the
correct configuration
 The proper glycosylation that are necessary for protien conformation,
localization by interacting with specific receptor and increase stability.

8. Eukaryotic Expression System
 Eukaryote expression vectors require sequence that encode for.
 Polyadenylation tail: creates a polyadenylation tail at the end of the
transcribed pre-mRNA that protects mRNA from exonucleases and
ensures transcriptional termination: stabilizers mRNA production.
 Minimal UTR length: UTRs contains a specific characteristics that may
impede transcriptional or translation, and thus the shortest UTRs or none
at are encoded for in optimal expression vectors.
 Kozak sequence: vectors should encode for a Kozak sequence in the m
RNA, which assembles the ribosome for translation of the m RNA.

9. Eukaryotic Expression System contd….
 Eukaryote expression vector require sequence that encode for.
 Polyadenylation tail: creates a polyadenylation tail at the end of the
transcribed pre-m RNA that protects the m RNA fromexonucleases and
ensure transcriptional and translational termination: stabilizers m RNA
production.
 Minimal UTR length: UTRs contains specific characteristics that may
impede transcription or translation, and thus the shortest UTRs or none at
all are encoded for in optimal expression vectors.
 Kozak sequence: vectors should encode encode for kozak sequence in the
m RNA, which assembles the ribosome for translation of the m RNA.

10. Element of of expression vector
 An expression vector has features that any vector may have, such as an
origin of replication, a selectable marker, and a suitable site for the
insertion of a gene like the multiple cloning site. The cloned gene may be
transferred from a specialized cloning vector to an expression vector,
although it is possible to clone directly into an expression vector. The
cloning process is normally performed in Escherichia coli. Vectors used for
protein production in organisms other than E.coli may have, in addition to
a suitable origin of replication for its propagation in E. coli, elements that
allow them to be maintained in another organism, and these vectors are
called shuttle vectors.

11.  The major feature of a eukaryotic expression vector are a promoter, a
multiple cloning site, DNA segment for termination and polyadenylation,
selectable marker, original of replication in E.coli and eukaryotic cell and
Ampr for marker in E.coli

12. Eukaryotic Expression System
 Saccharomyces cerevisiae
 Pichia pastoris
 Baculovirus-insect cell lines
 Mammalian systems

13. Elements of Expression Vector contd….
 An expression vector must have elements necessary for gene expression. These may
include a promoter, the correct translation initiation sequence such as a ribosomal
binding site and start codon, a termination codon, and a transcription termination
sequence. There are differences in the machinery for protein synthesis between
prokaryotes and eukaryotes, therefore the expression vectors must have the elements
for expression that are appropriate for the chosen host. For example, prokaryotes
expression vectors would have a Shine- Dalgarno sequence at its translation initiation
site for the binding of ribosomes, while eukaryotes expression vectors would contain the
Kozak consensus sequence.
 The promoter initiates the transcription and is therefore the point of control for the
expression of the cloned gene. The promoters used in expression vector are normally
inducible, meaning that protein synthesis is only initiated when required by the
introduction of an inducer such as IPTG. Gene expression however may also be
constitutive (i.e. protein is constantly expressed) in some expression vectors. Low level of
constitutive protein synthesis may occur even in expression vectors with tightly
controlled promoters.

14. Protein Tags
After the expression of the gene product, it is usually necessary to purify the expressed protein;
however, separating the protein of interest from the great majority of proteins of the host cell
can be a protracted process. To make this purification process easier, a purification tag may
be added to the cloned gene. This tag could be histidine (His) tag, other marker peptides, or a
fusion partners such as glutathione S-transferase or maltose-binding protein. Some of these
fusion partners may also help to increase the solubility of some expressed proteins. Other
fusion proteins such as green fluorescent protein may act as a reporter gene for the
identification of successful cloned genes, or they may be used to study protein expression in
cellular imaging.
OTHERS:
The expression vector is transformed or transfected into the host cell for protein synthesis.
Some expression vectors may have elements for transformation or the insertion of DNA into
the host chromosome, for example the vir genes for plant transformation, and integrase sites
for chromosomal integration. Some vectors may include targeting sequence that may target
the expressed protein to a specific location such as the periplasmic space of bacteria.

15. Expression/Production System
Different organisms may be used to express a gene's target protein, and the
expression vector used will therefore have elements specific for use in the
particular organism. The most commonly used organism for protein
production is the bacterium Escherichia coli. However, not all proteins can
be successfully expressed in E. coli, or be expressed with the correct form
of post-translational modifications such as glycosylations, and other
systems may therefore be used.

16. Bacteria
 The expression host of choice for the expression of many proteins is
Escherichia coli as the production of heterologous protein in E. coli is
relatively simple and convenient, as well as being rapid and cheap. A large
number of E. coli expression plasmids are also available for a wide variety
of needs. Other bacteria used for protein production include Bacillus
subtilis.
 Most heterologous proteins are expressed in the cytoplasm of E. coli.
However, not all proteins formed may be soluble in the cytoplasm, and
incorrectly folded proteins formed in cytoplasm can form insoluble
aggregates called inclusion bodies. Such insoluble proteins will require
refolding, which can be an involved process and may not necessarily
produce high yield

17. FIG- EXPRESSION VECTOR

18.  Proteins which have disulphide bonds are often not able to fold correctly due to the reducing
environment in the cytoplasm which prevents such bond formation, and a possible solution is
to target the protein to the periplasmic space by the use of an N-terminal signal sequence.
 Another possibility is to manipulate the redox environment of the cytoplasm. Other more
sophisticated systems are also being developed; such systems may allow for the expression of
proteins previously thought impossible in E. coli, such as glycosylated proteins.
 The promoters used for these vector are usually based on the promoter of the lac operon or
the T7 promoter, and they are normally regulated by the lac operator. These promoters may
also be hybrids of different promoters, for example, the Tac-Promoter is a hybrid of trp and lac
promoters. Note that most commonly used lac or lac-derived promoters are based on the
lacUV5 mutant which is insensitive to catabolite repression. This mutant allows for expression of
protein under the control of the lac promoter when the growth medium contains glucose since
glucose would inhibit gene expression if wild-type lac promoter is used. Presence of glucose
nevertheless may still be used to reduce background expression through residual inhibition in
some systems.
 Examples of E. coli expression vectors are the pGEX series of vectors where glutathione S-
transferase is used as a fusion partner and gene expression is under the control of the tac
promoter, and the pET series of vectors which uses a T7 promoter.

19. Bacteria Contd…
 It is possible to simultaneously express two or more different
proteins in E. coli using different plasmids. However, when 2 or
more plasmids are used, each plasmid needs to use a different
antibiotic selection as well as a different origin of replication,
otherwise one of the plasmids may not be stably maintained.
Many commonly used plasmids are based on the ColE1 replicon
and are therefore incompatible with each other; in order for a
ColE1-based plasmid to coexist with another in the same cell,
the other would need to be of a different replicon, e.g. a p15A
replicon-based plasmid such as the pACYC series of plasmids.
Another approach would be to use a single two-cistron vector
or design the coding sequences in tandem as a bi- or poly-
cistronic construct.

20. Yeast
 A yeast commonly used for protein production is Pichia pastoris. Examples of yeast expression
vector in Pichia are the pPIC series of vectors, and these vectors use the AOX1 promoter
which is inducible with methanol. The plasmids may contain elements for insertion of foreign
DNA into the yeast genome and signal sequence for the secretion of expressed protein.
Proteins with disulphide bonds and glycosylation can be efficiently produced in yeast. Another
yeast used for protein production is Kluyveromyces lactis and the gene is expressed, driven by
a variant of the strong lactase LAC4 promoter.
 Saccharomyces cerevisiae is particularly widely used for gene expression studies in yeast, for
example in yeast two-hybrid system for the study of protein-protein interaction. The vectors
used in yeast two-hybrid system contain fusion partners for two cloned genes that allow the
transcription of a reporter gene when there is interaction between the two proteins expressed
from the cloned genes.
 Baculovirus is normally used for production of glycoproteins, although the glycosylations may
be different from those found in vertebrates. In general, it is safer to use than mammalian
virus as it has a limited host range and does not infect vertebrates without modifications.

22. YAC Cloning System
 A YAC is designed to clone a large segments of DNA (100kb), which is then
maintained as a separate chromosome in the host yeast cell.
 It is highly stable and has been used for the physical mapping of human
genomic libraries.
 It has a sequences that act as ARS for replication, centromere for cell
division, and telomere for stability.
 To date, they have not been used as expression systems for the
commercial production.

23. Intercellular Production in Yeast
 Human Cu/ZnSOD c DNA was cloned between the promoter and
termination-polyadenylation sequence of the yeast GAPD gene and
subsequently used to transform LEU mutant host cell.

24. Secretion of Heterologous Proteins
 Proteins may also be production for secretion.
 In this system, any glycosylated protein is selected
 The coding sequence of recombinant protein must be cloned downstream of a
leader sequence, the appropriate posttranslational modifications occur, and an
active recombinant Protein in secreted.
 The leader peptide is removed by endoprotease that recognizes the Lys-Arg.
 For Example-a properly processed and activity from the protein hirudine;
coding sequence to allow expression that is cleaved away in processing.
 Producing a recombinant in yeast rather than in bacteria is to ensure the
proper folding.

25. Difference Between Cloning Vector &
Expression Vector

26. Secretion of heterologous proteins

27. Cell- Free System
 E. coli cell lysate containing the cellular components required for
transcription and translation are used in this in vitro method of protein
production. The advantage of such system is that protein may be
produced much faster than those produced in vivo since it does not
require time to culture the cells, but it is also more expensive. Vectors used
for E. coli expression can be used in this system although specifically
designed vectors for this system are also available. Eukaryotic cell extracts
may also be used in other cell-free systems, for example, the wheat germ
cell-free expression systems. Mammalian cell-free systems have also been
produced.

28. Vector Design
 Generalized mammalian expression vector.
 The MCS and SMG are under the control of eukaryotic promoter, polyadenylation, and
terminal sequences.
 An intron enhance the production of heterologous protein.
 The AMPr gene is used for selecting transformed E.COLI.
 For the best results, a gene of interest must be equipped with translation control
sequence
 A gene of interest with a various sequences that enhance translation and facilitate both
secretion and purification.
 A Kozak sequence, specific sequence surrounding the AUG start codon, signal
sequence, protein affinity tag for purification, protelaolytic cleavage site,and stop codon.
 The 5’ & 3’ UTR increase the efficiency of translation and contribute to mRNA stability.

29. Two Vector Expression System

30. Two Vector Expression System contd….

31. Application
 LABORATORY
 Expression vector in an expression host is now the usual method used in
laboratories to produce proteins for research. Most proteins are produced
in E. coli, but for glycosylated proteins and those with disulphide bonds,
yeast, Baculovirus and mammalian systems may be used.

32. Production of Peptide and Protein
Pharmaceuticals
 Most protein pharmaceuticals are now produced through recombinant DNA
technology using expression vectors. These peptide and protein
pharmaceuticals may be hormones, vaccines, antibiotics, antibodies, and
enzymes. The first human recombinant protein used for disease management,
insulin, was introduced in 1982. Biotechnology allows these peptide and
protein pharmaceuticals, some of which were previously rare or difficult to
obtain, to be produced in large quantity. It also reduces the risks of
contaminants such as host viruses, toxins and prions. Examples from the past
include prion contamination in growth hormone extracted from pituitary
glands harvested from human cadavers, which caused Creutzfeldt–Jakob
disease in patients receiving treatment for dwarfism, and viral contaminants in
clotting factor VIII isolated from human blood that resulted in the transmission
of viral diseases such as hepatitis and AIDS. Such risk is reduced or removed
completely when the proteins are produced in non-human host cells.

33. Reference
https://www.cusabio.com/c-20273.html
https://en.wikipedia.org/wiki/Expression_vector