An expression vector, otherwise known as an expression construct, is usually a
plasmid or virus designed for protein 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 plasmid 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 protein may be tightly controlled and 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 expression, 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.
Elements of expression vectors 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 such as 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, and vectors used for protein expression 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.
= Elements for expression= An expression vector must have elements
necessary for protein expression. These may include a strong promoter, the
correct translation initiation sequence such as a ribosomal binding site and
start codon, a strong 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 is 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. Protein expression
however may also be constitutive in some expression vectors. Low level of
constitutive protein synthesis may occur even in expression vectors with tightly
controlled promoters. = 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 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. = 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
insertion. Some vectors may include targeting
sequence that may target the expressed protein to a specific location such as
the periplasmic space of bacteria. Expression systems
Different organisms may be used to express a target protein, the expression
vector used therefore will have elements specific for use in the particular
organism. The most commonly used organism for protein expression 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. = Bacterial=
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 suitable for a wide
variety of needs. Other bacteria used for protein expression 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 body. Such insoluble proteins will
require refolding, which can be an involved process and may not necessarily
produce high yield. Proteins which have disulphide bondss are often not able to
fold correctly due to the oxidizing 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.
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
protein 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 protein expression is under the control of the tac promoter,
and the pET series of vectors which uses a T7 promoter.
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 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. = Yeast=
A yeast commonly used for protein expression 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 expression is Kluyveromyces lactis and
the protein is expressed driven by a variant of the strong lactase LAC4
promoter. Saccharomyces cerevisiae is particularly
widely used for protein 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= Baculovirus, a rod-shaped virus which
infect insect cells, is used as the expression vector in this system. Insect
cell lines derived from Lepidopterans, such as Spodoptera frugiperda, are used
as host. The shuttle vector is called bacmid, and protein expression is under
the control of a strong promoter pPolh. Baculovirus has also been used with
mammalian cell lines in the BacMam system.
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.
= Plant= Many plant expression vectors are based
on the Ti plasmid of Agrobacterium tumefaciens. In these expression
vectors, DNA to be inserted into plant is cloned into the T-DNA, a stretch of
DNA flanked by a 25-bp direct repeat sequence at either end, and which can
integrate into the plant genome. The T-DNA also contains the selectable
marker. The Agrobacterium provides a mechanism for transformation,
integration of into the plant genome, and the promoters for its vir genes may
also be used for the cloned genes. Other plant viruses may be used as
vectors since Agrobacterium does not work for all plants. Examples of plant
virus used are the tobacco mosaic virus, potato virus X, and cowpea mosaic virus.
The protein may be expressed as a fusion to the coat protein of the virus and is
displayed on the surface of assembled viral particles, or as an unfused
protein that accumulates within the plant. Expression in plant using plant
vectors is often constitutive, and a commonly used constitutive promoter in
plant expression vectors is the cauliflower mosaic virus 35S promoter.
= Mammalian= Mammalian expression vectors offer
considerable advantages for the expression of mammalian proteins over
bacterial expression systems - proper folding, post-translational
modifications, and relevant enzymatic activity. It may also be more desirable
than other eukaryotic non-mammalian systems whereby the proteins expressed
may not contain the correct glycosylations. It is of particular use
in producing membrane-associating proteins that require chaperones for
proper folding and stability as well as containing numerous post-translational
modifications. The downside, however, is the low yield of product in comparison
to prokaryotic vectors as well as the costly nature of the techniques
involved. Its complicated technology, and potential contamination with animal
viruses of mammalian cell expression have also placed a constraint its use in
large-scale industrial production. Cultured mammalian cell lines such as
the Chinese hamster ovary, HEK, HeLa, and COS cell lines may be used to
produce protein. Vectors are transfected into the cells and the DNA may be
integrated into the genome by homologous recombination in the case of stable
transfection, or the cells may be transiently transfected. Examples of
mammalian expression vectors include the adenoviral vectors, the pSV and the pCMV
series of plasmid vectors, vaccinia and retroviral vectors, as well as
baculovirus. The promoters for cytomegalovirus and SV40 are commonly
used in mammalian expression vectors to drive protein expression. Non-viral
promoter, such as the elongation factor-1 promoter, is also known.
= Cell-free systems= E. coli cell lysate containing the
cellular components required for transcription and translation are used
in this in vitro method of protein expression. 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. Applications
= Laboratory use= 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. = 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 was insulin and it 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 amount.
It also reduces the risks of contaminants such as host viruses,
toxins and prions. For example, growth hormone extracted from pituitary glands
harvested from human cadavers had caused Creutzfeldt–Jakob disease in patients
receiving treatment for dwarfism due to prion contamination, and viral
contaminants in clotting factor VIII isolated from human blood had resulted
in the transmission of viral diseases such as hepatitis and AIDS. Such risk is
reduced or removed completely when these proteins are produced in non-human
cell-lines. = Transgenic plant and animals=
In recent years, expression vectors have been used to introduce specific genes
into plants and animals to produce transgenic organisms, for example in
agriculture it is used to produce transgenic plants. Expression vectors
have been used to introduce a vitamin A precursor, beta-carotene, into rice
plants. This product is called golden rice. This process has also been used to
introduce a gene into plants that produces an insecticide, called Bacillus
thuringiensis toxin or Bt toxin which reduces the need for farmers to apply
insecticides since it is produced by the modified organism. In addition
expression vectors are used to extend the ripeness of tomatoes by altering the
plant so that it produces less of the chemical that causes the tomatoes to
rot. There have been controversies over using expression vectors to modify crops
due to the fact that there might be unknown health risks, possibilities of
companies patenting certain genetically modified food crops, and ethical
concerns. Nevertheless, this technique is still being used and heavily
researched. Transgenic animals have also been
produced to study animal biochemical processes and human diseases, or used to
produce pharmaceuticals and other proteins. They may also be engineered to
have advantageous or useful traits. Green fluorescent protein is sometimes
used as tags which results in animal that can fluoresce, and this have been
exploited commercially to produce the fluorescent GloFish.
= Gene therapy= Gene therapy is a promising treatment
for a number of diseases where a "normal" gene carried by the vector is
inserted into the genome, to replace an "abnormal" gene or supplement the
expression of particular gene. Viral vectors are generally used but other
nonviral methods of delivery are being developed. The treatment is still a
risky option due to the viral vector used which can cause ill-effects, for
example giving rise to insertional mutation that can result in cancer.
However, there have been promising results.
See also Cloning vector
References External links
GST Gene Fusion System Handbook