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===Elements for expression===
{{further|Transcription (genetics)|Translation (biology)}}
An expression vector must have elements necessary for gene expression. These may include a [[Promoter (genetics)|promoter]], the correct translation initiation sequence such as a [[ribosomal binding site]] and [[start codon]], a [[termination codon]], and a [[Terminator (genetics)|transcription termination sequence]].<ref>{{cite book |title=Principles of Gene Manipulation |chapter-url=https://archive.org/details/principlesofgene00oldr |chapter-url-access=registration |chapter=Chapter 8: Expression E. coli of cloned DNA molecules |
The [[Promoter (genetics)|promoter]] initiates the [[Transcription (genetics)|transcription]] and is therefore the point of control for the expression of the cloned gene. The promoters used in expression vector are normally [[Enzyme induction and inhibition|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.
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===Protein tags===
{{main|Protein tag}}
After the expression of the gene product, it may be 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 [[Protein tag|purification tag]] may be added to the cloned gene. This tag could be [[Polyhistidine-tag|histidine (His) tag]], other marker peptides, or a [[fusion protein|fusion partners]] such as [[glutathione S-transferase]] or [[maltose-binding protein]].<ref>{{cite journal |title= Overview of Affinity Tags for Protein Purification |author1=Michelle E. Kimple |author2=Allison L. Brill |author3=Renee L. Pasker |journal=Current Protocols in Protein Science |date=24 September 2013 | volume=73|issue=Unit-9.9 |pages=9.9.1–9.9.23 |pmid= 24510596 |pmc=4527311 |doi= 10.1002/0471140864.ps0909s73 |isbn={{Format ISBN|9780471140863}} }}</ref> 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 [[Live cell imaging|cellular imaging]].<ref>{{cite journal |title=Design and Use of Fluorescent Fusion Proteins in Cell Biology |author=Erik Snapp |journal=Current Protocols in Cell Biology |volume=27 |pages=21.4.1–21.4.13 |date=July 2005
===Other Elements===
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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.<ref>{{cite book |series=Methods in Enzymology |year= 2009 |volume= 463 |pages=259–82 |doi= 10.1016/S0076-6879(09)63017-2 |title=Refolding solubilized inclusion body proteins |author= Burgess RR |pmid=19892177|isbn= {{Format ISBN|9780123745361}} }}</ref> 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 peptide|signal sequence]]. Another possibility is to manipulate the redox environment of the cytoplasm.<ref>{{cite journal |title=SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm |
The promoters used for these vector are usually based on the promoter of the [[lac operon|''lac'' operon]] or the [[T7 phage|T7]] promoter,<ref>{{cite journal |vauthors=Dubendorff JW, Studier FW |title=Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor |journal=Journal of Molecular Biology |year=1991 |volume=219 |issue=1 |pages=45–59 |pmid=1902522 |doi=10.1016/0022-2836(91)90856-2}}</ref> and they are normally regulated by the ''lac'' [[Operator (biology)|operator]]. These promoters may also be hybrids of different promoters, for example, the [[Tac-Promoter]] is a hybrid of [[trp operon|''trp'']] and ''lac'' promoters.<ref>{{cite journal |
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,<ref>{{cite journal |title=Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase |vauthors=Smith DB, Johnson KS |journal=Gene |year=1988 |volume=67|issue=1 |pages=31–40|pmid=3047011 |doi=10.1016/0378-1119(88)90005-4}}</ref><ref>{{cite web |title=GST Gene Fusion System |url=http://wolfson.huji.ac.il/purification/PDF/Tag_Protein_Purification/GST/PHARMACIA_GST_Gene_Fusion_System_Handbook.pdf |work=Amersham Pharmacia biotech }}</ref><ref>{{cite web |url=http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences/products/AlternativeProductStructure_16996/28954653 |title=pGEX Vectors |publisher= GE Healthcare Lifesciences }}</ref> and the pET series of vectors which uses a [[T7 phage|T7]] promoter.<ref>{{cite web |url= http://lifeserv.bgu.ac.il/wb/zarivach/media/protocols/Novagen%20pET%20system%20manual.pdf |title=pET System manual |work=Novagen }}</ref>
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===Plant===
Many plant expression vectors are based on the [[Ti plasmid]] of ''[[Agrobacterium tumefaciens]]''.<ref>{{cite journal |title=Techniques in plant molecular biology--progress and problems |vauthors=Walden R, Schell J |journal=European Journal of Biochemistry |year= 1990 |volume=192 |issue=3 |pages=563–76 |pmid= 2209611|doi=10.1111/j.1432-1033.1990.tb19262.x |doi-access= }}</ref> In these expression vectors, DNA to be inserted into plant is cloned into the [[T-DNA Binary system|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 (genetics)|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.<ref>{{cite book |url=https://books.google.com/books?id=mpc02lNJRs8C&pg=PT629 |title=Principles of Plant Genetics and Breeding|author= George Acquaah |date=16 August 2012|publisher= John Wiley & Sons Inc |isbn={{Format ISBN|9781118313695}} }}</ref>
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]].<ref>{{cite journal |title= Use of viral vectors for vaccine production in plants |author1=M Carmen Cañizares |author2=Liz Nicholson |author3=George P Lomonossoff |journal=Immunology and Cell Biology |year=2005 |volume=83 |issue=3 |pages= 263–270 |doi=10.1111/j.1440-1711.2005.01339.x |pmid=15877604 |pmc=7165799 }}</ref> 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,<ref>{{cite web |title=How Do You Make A Transgenic Plant? |url=http://cls.casa.colostate.edu/transgeniccrops/how.html |work=Department of Soil and Crop Sciences at Colorado State University }}</ref> and a commonly used constitutive promoter in plant expression vectors is the [[cauliflower mosaic virus]] (CaMV) 35S promoter.<ref>{{cite journal |author1=Fütterer J. |author2=Bonneville J. M. |author3=Hohn T |title=Cauliflower mosaic virus as a gene expression vector for plants |journal=Physiologia Plantarum
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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 on its use in large-scale industrial production.<ref name="mammalian">{{cite journal |title=Gene Expression in Mammalian Cells and its Applications|author= Kishwar Hayat Khan |journal= Adv Pharm Bull. |year= 2013 |volume= 3 |issue=2 |pages= 257–263 |pmid=24312845 |pmc=3848218 | doi= 10.5681/apb.2013.042 }}</ref>
Cultured mammalian cell lines such as the [[Chinese hamster ovary cell|Chinese hamster ovary (CHO)]], [[COS cells|COS]], including human cell lines such as [[HEK cell|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,<ref>{{cite book |year= 1992 |volume=158 |pages=39–66 |author=Berkner KL |title= Viral Expression Vectors |chapter= Expression of Heterologous Sequences in Adenoviral Vectors |series= Current Topics in Microbiology and Immunology |pmid=1582245 |doi= 10.1007/978-3-642-75608-5_3 |isbn= 978-3-642-75610-8 }}</ref> the pSV and the pCMV series of plasmid vectors, [[vaccinia]] and [[retroviral]] vectors,<ref>{{cite journal |journal=Clin Microbiol Rev |year=1990 |volume= 3 |issue=2|pages= 153–170 |pmc=358149|title=Vaccinia virus vectors: new strategies for producing recombinant vaccines |author=Hruby, DE |pmid=2187593 |doi=10.1128/cmr.3.2.153}}</ref> as well as baculovirus.<ref name="Kost2002">{{cite journal|pmid=11906750|doi=10.1016/S0167-7799(01)01911-4|title=Recombinant baculoviruses as mammalian cell gene-delivery vectors|year=2002|last1=Kost|first1=T|journal=Trends in Biotechnology|volume=20|issue=4|pages=173–180|last2=Condreay|first2=JP}}</ref> 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.<ref>{{cite journal |journal=Gene |year=1990 |volume=91 |issue=2 |pages=217–23 |title=Use of the human elongation factor 1 alpha promoter as a versatile and efficient expression system |
===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.<ref>{{cite book |title=Current Protocols in Protein Science |volume= Chapter 5 |pages= 5.18.1–5.18.18 |year= 2006 |chapter=Chapter 5:Unit 5.18. Wheat Germ Cell-Free Expression System for Protein Production |doi= 10.1002/0471140864.ps0518s44 |pmid= 18429309 |vauthors=Vinarov DA, Newman CL, Tyler EM, Markley JL, Shahan MN |isbn= {{Format ISBN|9780471140863}}|s2cid= 12057689 }}</ref> Mammalian cell-free systems have also been produced.<ref>{{cite book |year= 2015 |volume=1261 |pages=129–40 | doi= 10.1007/978-1-4939-2230-7_7 |
==Applications==
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===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|Bacillus thuringiensis toxin]] or [[Bacillus thuringiensis|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.<ref>{{Cite web |url=http://www.bionetonline.org/english/content/ff_cont3.htm |title=bionetonline.org |access-date=2010-06-12 |archive-url=https://web.archive.org/web/20100617043538/http://www.bionetonline.org/english/content/ff_cont3.htm |archive-date=2010-06-17
[[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]].
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