Editing Expression vector (section)

==Expression/Production systems==
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===
[[File:pGEX-3X cloning vector.png|thumb|An example of a bacterial expression vector is the pGEX-3x plasmid]]
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 |author= Burgess RR |title= Guide to Protein Purification, 2nd Edition |chapter= Chapter 17 Refolding Solubilized Inclusion Body Proteins |pmid=19892177|isbn= 978-0-12-374536-1 }}</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 |author=Julie Lobstein |author2=Charlie A Emrich |author3=Chris Jeans |author4=Melinda Faulkner |author5=Paul Riggs |author6=Mehmet Berkmen |journal=Microbial Cell Factories|date= 2012|volume= 11|page= 56 |pmc=3526497 |pmid=22569138 |doi=10.1186/1475-2859-11-56 |doi-access=free }}</ref> 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.<ref>{{cite journal |title=N-linked glycosylation in Campylobacter jejuni and its functional transfer into E. coli |vauthors=Wacker M, Linton D, Hitchen PG, Nita-Lazar M, Haslam SM, North SJ, Panico M, Morris HR, Dell A, Wren BW, Aebi M |journal=Science |volume=298 |issue=5599 |pages=1790–1793 |year=2002 |pmid=12459590 |doi=10.1126/science.298.5599.1790|bibcode=2002Sci...298.1790W }}</ref><ref>{{cite journal |title=Industrial production of recombinant therapeutics in Escherichia coli and its recent advancements |vauthors=Huang CJ, Lin H, Yang X |journal=J Ind Microbiol Biotechnol |volume=39 |issue=3 |pages=383–99 |year=2012 |pmid=22252444 |doi=10.1007/s10295-011-1082-9|s2cid=15584320 |doi-access=free }}</ref><ref>{{cite journal |title=Recombinant protein expression in Escherichia coli: advances and challenges|author1=Germán L. Rosano1 |author2=Eduardo A. Ceccarelli |journal=Frontiers in Microbiology |date= 2014|volume= 5 |page= 172 |pmid= 24860555 |pmc=4029002 |doi=10.3389/fmicb.2014.00172|doi-access=free }}</ref>

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 |vauthors=deBoer HA, Comstock LJ, Vasser M |year=1983|title= The tac promoter: a functional hybrid derived from trp and lac promoters |journal= Proceedings of the National Academy of Sciences USA |volume=80 |pages=21–25 |pmid=6337371 |issue=1 |pmc=393301 |doi=10.1073/pnas.80.1.21|bibcode=1983PNAS...80...21D|doi-access=free}}</ref> Note that most commonly used ''lac'' or ''lac''-derived promoters are based on the [[LacUV5|''lac''UV5]] 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.<ref>{{cite journal |vauthors=Silverstone AE, Arditti RR, Magasanik B |title= Catabolite-insensitive revertants of lac promoter mutants |year=1970 |journal= Proceedings of the National Academy of Sciences USA |volume=66 |issue=3 |pages=773–9 |pmid=4913210 |pmc=283117 |doi=10.1073/pnas.66.3.773|bibcode= 1970PNAS...66..773S |doi-access= free }}</ref> Presence of glucose nevertheless may still be used to reduce background expression through residual inhibition in some systems.<ref>{{cite journal |url=http://wolfson.huji.ac.il/expression/procedures/bacterial/Glucose%20supression.pdf |title=Use of glucose to control basal expression in the pET System |author1=Robert Novy |author2=Barbara Morris |journal=InNovations |number=13 |pages=6–7 }}</ref>

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 |access-date=2013-10-11 |archive-date=2016-11-13 |archive-url=https://web.archive.org/web/20161113231639/http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences/products/AlternativeProductStructure_16996/28954653 |url-status=dead }}</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 |access-date=2012-12-11 |archive-date=2019-08-19 |archive-url=https://web.archive.org/web/20190819055404/http://lifeserv.bgu.ac.il/wb/zarivach/media/protocols/Novagen%20pET%20system%20manual.pdf |url-status=dead }}</ref>

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.<ref>{{cite book |title=E. coli Plasmid Vectors: Methods and Applications |author1=Nicola Casali |author2=Andrew Preston |series = Methods in Molecular Biology|volume=235 |page=22 |isbn=978-1-58829-151-6 |date=2003-07-03 }}</ref> 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.<ref>{{cite web |url=http://www.embl.de/pepcore/pepcore_services/cloning/cloning_methods/dicistronic_cloning/index.html |title=Cloning Methods - Di- or multi-cistronic Cloning |work=EMBL }}</ref><ref>{{cite journal |title=Translation of a synthetic two-cistron mRNA in Escherichia coli |vauthors=Schoner BE, Belagaje RM, Schoner RG |journal= Proc Natl Acad Sci U S A |year=1986 |volume=83 |issue=22|pages=8506–10 |pmid= 3534891 |pmc=386959 |doi=10.1073/pnas.83.22.8506|bibcode=1986PNAS...83.8506S |doi-access=free }}</ref>

===Yeast===
A yeast commonly used for protein production is ''[[Pichia pastoris]]''.<ref>{{cite journal |title= Recombinant protein expression in Pichia pastoris |vauthors=Cregg JM, Cereghino JL, Shi J, Higgins DR |journal=Molecular Biotechnology |year=2000 |volume=16 |issue=1 |pages=23–52 |pmid= 11098467 |doi=10.1385/MB:16:1:23|s2cid=35874864 |doi-access=free }}</ref> 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]].<ref>{{cite web |url=http://tools.invitrogen.com/content/sfs/brochures/B-067202_Pichia_Flyer.pdf |title=Pichia pastoris Expression System |work=Invitrogen }}</ref> 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.<ref>{{cite web |url=https://www.neb.com/~/media/Catalog/All-Products/B1A99D5EBC6E45B3B876E40A8ECCED3F/Datacards%20or%20Manuals/manualE1000.pdf |title=K. lactis Protein Expression Kit |work=New England BioLabs Inc. |access-date=2013-03-20 |archive-date=2016-03-04 |archive-url=https://web.archive.org/web/20160304185904/https://www.neb.com/~/media/Catalog/All-Products/B1A99D5EBC6E45B3B876E40A8ECCED3F/Datacards%20or%20Manuals/manualE1000.pdf |url-status=dead }}</ref>

''[[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.<ref>{{cite journal |vauthors=Fields S, Song O |title=A novel genetic system to detect protein-protein interactions |journal=Nature |volume=340 |issue=6230 |pages=245–6 |year=1989 |pmid=2547163 |doi=10.1038/340245a0 |bibcode=1989Natur.340..245F |s2cid=4320733 }}</ref> 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.<ref>{{cite web|title=The Baculovirus Expression Vector System (BEVS)|author=Mckenzie, Samuel|date=February 26, 2019|website=news-medical.net|url=https://www.news-medical.net/life-sciences/The-Baculovirus-Expression-Vector-System-(BEVS).aspx}}</ref> Insect cell lines derived from [[Lepidopteran]]s (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.<ref>{{Cite journal|last=HINK|first=W. F.|date=1970-05-02|title=Established Insect Cell Line from the Cabbage Looper, Trichoplusia ni|journal=Nature|language=en|volume=226|issue=5244|pages=466–467|doi=10.1038/226466b0|pmid=16057320|issn=1476-4687|bibcode=1970Natur.226..466H|s2cid=4225642}}</ref><ref>{{cite journal |vauthors=Zheng GL, Zhou HX, Li CY |date=2014|title= Serum-free culture of the suspension cell line QB-Tn9-4s of the cabbage looper, Trichoplusia ni, is highly productive for virus replication and recombinant protein expression|journal= Journal of Insect Science |volume=14|issue=1|page=24 |pmid= 25373171 |pmc=4199540|doi=10.1093/jis/14.1.24}}</ref> The [[shuttle vector]] is called bacmid, and gene expression is under the control of a strong promoter pPolh.<ref>{{cite web |url=http://tools.invitrogen.com/content/sfs/manuals/bevtest.pdf |title=Guide to Baculovirus Expression Vector Systems (BEVS) and Insect Cell Culture Techniques|work=Invitrogen }}</ref> Baculovirus has also been used with mammalian cell lines in the [[BacMam]] system.<ref name="Kost2002"/>

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]]''.<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=978-1-118-31369-5 }}</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 |access-date=2013-02-06 |archive-date=2013-01-21 |archive-url=https://web.archive.org/web/20130121061854/http://cls.casa.colostate.edu/TransgenicCrops/how.html |url-status=dead }}</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
|volume = 79 |issue = 1 |pages = 154–157 |date= May 1990 |doi= 10.1111/j.1399-3054.1990.tb05878.x |bibcode=1990PPlan..79..154F }}</ref><ref>{{cite journal |title=The Cauliflower Mosaic Virus 35S Promoter: Combinatorial Regulation of Transcription in Plants |vauthors=Benfey PN, Chua NH |journal=Science |year=1990 |volume=250|issue=4983 |pages=959–66 |pmid=17746920 |url=http://www.sciencemag.org/site/feature/data/plants2001/PDFs/250-4983-959.pdf |doi=10.1126/science.250.4983.959|bibcode=1990Sci...250..959B |s2cid=35471862 }}</ref>

===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 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 |vauthors=Kim DW, Uetsuki T, Kaziro Y, Yamaguchi N, Sugano S |pmid =2210382 |doi=10.1016/0378-1119(90)90091-5}}</ref>

===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= 978-0-471-14086-3|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 |vauthors=Brödel AK, Wüstenhagen DA, Kubick S |chapter=Cell-Free Protein Synthesis Systems Derived from Cultured Mammalian Cells |title = Structural Proteomics|pmid=25502197 |series= Methods in Molecular Biology |isbn= 978-1-4939-2229-1 }}</ref>