Expression vector: Difference between revisions

Revision as of 22:16, 18 August 2023

Revision as of 22:00, 1 September 2023

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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 journalbook |journalseries=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= 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 |authors=Julie Lobstein, Charlie A Emrich, Chris Jeans, Melinda Faulkner, Paul Riggs, and 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 |author=deBoer H. A., Comstock, L. J., 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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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 journal |journal=Current Topics in Microbiology and Immunologybook |year= 1992 |volume=158 |pages=39–66 |author=Berkner KL |title= Viral Expression Vectors |chapter= Expression of heterologousHeterologous sequencesSequences in adenoviralAdenoviral vectorsVectors |authorseries=Berkner KLCurrent 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 |author=Kim DW1, 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= 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 |chapter=Cell-free protein synthesis systems derived from cultured mammalian cells |author=Brödel AK1, 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>

==Applications==