Expression vector: Difference between revisions

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An '''expression vector''', otherwise known as an '''expression construct''', is usually a [[plasmid]] or virus designed for [[gene expression]] in cells. The [[vector (molecular biology)|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]] [[Genetic code|encoded]] by the gene. Expression vectors are the basic tools in [[biotechnology]] for the [[protein production|production of proteins]].

The [[Vector (molecular biology)|vector]] is engineered to contain regulatory sequences that act as [[Enhancer (genetics)|enhancer]] and [[Promoter (biology)|promoter]] regions and lead to efficient transcription of the gene carried on the expression vector.<ref>[http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/in-vitro-genetics/expression-vectors.html sci.sdsu.edu]</ref> 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 [[Translation (biology)|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 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]].

==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 |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 |article-number=753 |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>

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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=No.: 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===

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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===