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.
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 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. 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. 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.
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 Baculovirus, a rod-shaped virus which infects insect cells, is used as the expression vector in this system. Insect cell lines derived from
Lepidopterans (moths and butterflies), such as
Spodoptera frugiperda, are used as host. A cell line derived from the
cabbage looper is of particular interest, as it has been developed to grow fast and without the expensive serum normally needed to boost cell growth. The
shuttle vector is called bacmid, and gene expression is under the control of a strong promoter pPolh. Baculovirus has also been used with mammalian cell lines in the
BacMam system. 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. Concerns over the transfer of bacterial or viral genetic material into the plant however have led to the development of vectors called intragenic vectors whereby functional equivalents of plant genome are used so that there is no transfer of genetic material from an alien species into the plant. Plant viruses may be used as vectors since the
Agrobacterium method does not work for all plants. Examples of plant virus used are the
tobacco mosaic virus (TMV),
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 (CaMV) 35S promoter. Cultured mammalian cell lines such as the
Chinese hamster ovary (CHO),
COS, including human cell lines such as
HEK and
HeLa 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 (CMV) and
SV40 are commonly used in mammalian expression vectors to drive gene expression. Non-viral promoter, such as the elongation factor (EF)-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 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. ==Applications==