Content deleted Content added
m Reverted edits by 2405:201:3015:C80B:49F7:399E:34BE:7388 (talk) (HG) (3.4.10) |
Citation bot (talk | contribs) Added article-number. | Use this bot. Report bugs. | Suggested by Headbomb | Linked from Wikipedia:WikiProject_Academic_Journals/Journals_cited_by_Wikipedia/Sandbox2 | #UCB_webform_linked 213/741 |
||
| (17 intermediate revisions by 10 users not shown) | |||
Line 1:
{{Short description|Virus or plasmid designed for gene expression in cells}}
[[File:PET28a-T7-lacO-GFP.svg|thumb|A bacterial expression vector for expressing [[green fluorescent protein]] from the [[T7 RNA polymerase|T7 promoter]].]]
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]]
==Elements==
Line 11 ⟶ 12:
===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.
Line 17 ⟶ 18:
===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 |
===Other Elements===
Line 31 ⟶ 32:
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
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=
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=
===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.
Line 50 ⟶ 51:
===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=
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
===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=
==Applications==
Line 73 ⟶ 74:
===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]].
Line 89 ⟶ 90:
==External links==
*[http://www.gelifesciences.com/handbooks GST Gene Fusion System Handbook] {{Webarchive|url=https://web.archive.org/web/20081205061748/http://www.gelifesciences.com/handbooks |date=2008-12-05 }}
{{DEFAULTSORT:Expression Vector}}
[[Category:Genetics techniques]]
[[Category:Molecular biology]]
[[Category:Biotechnology]]
| |||