Expression vector: Difference between revisions

Revision as of 14:53, 10 September 2023

Revision as of 08:48, 23 October 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 book |series=Methods in Enzymology |year= 2009 |volume= 463 |pages=259–82 |doi= 10.1016/S0076-6879(09)63017-2 |author= Burgess RR |title=Refolding solubilizedGuide inclusionto bodyProtein proteinsPurification, 2nd Edition |authorchapter= BurgessChapter RR17 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>