Editing Expression vector (section)

==Elements==

An expression vector has features that any [[Vector (molecular biology)|vector]] may have, such as an [[origin of replication]], a [[selectable marker]], and a suitable site for the insertion of a gene like the [[multiple cloning site]]. The cloned gene may be transferred from a specialized [[cloning vectors|cloning vector]] to an expression vector, although it is possible to clone directly into an expression vector. The cloning process is normally performed in ''[[Escherichia coli (molecular biology)|Escherichia coli]]''. Vectors used for protein production in organisms other than ''E.coli'' may have, in addition to a suitable origin of replication for its propagation in ''E. coli'', elements that allow them to be maintained in another organism, and these vectors are called [[shuttle vector]]s.

===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 |author=RW Old |author2=SB Primrose |year=1994 |publisher=Blackwell Scientific Publications |isbn=978-0-632-03712-4 }}</ref> There are differences in the machinery for protein synthesis between prokaryotes and eukaryotes, therefore the expression vectors must have the elements for expression that are appropriate for the chosen host.  For example, prokaryotes expression vectors would have a [[Shine-Dalgarno sequence]] at its translation initiation site for the binding of ribosomes, while eukaryotes expression vectors would contain the [[Kozak consensus sequence]].

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.

===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 |author1=Michelle E. Kimple |author2=Allison L. Brill |author3=Renee L. Pasker |journal=Current Protocols in Protein Science |date=24 September 2013 | volume=73|issue=Unit-9.9 |pages=9.9.1–9.9.23 |pmid= 24510596 |pmc=4527311 |doi= 10.1002/0471140864.ps0909s73 |isbn=978-0-471-14086-3 }}</ref> Some of these fusion partners may also help to increase the solubility of some expressed proteins. Other fusion proteins such as [[green fluorescent protein]] may act as a [[reporter gene]] for the identification of successful cloned genes, or they may be used to study protein expression in [[Live cell imaging|cellular imaging]].<ref>{{cite journal |title=Design and Use of Fluorescent Fusion Proteins in Cell Biology |author=Erik Snapp |journal=Current Protocols in Cell Biology |volume=27 |pages=21.4.1–21.4.13 |date=July 2005 | doi= 10.1002/0471143030.cb2104s27 |pmc=2875081 |pmid =18228466}}</ref><ref>{{cite journal |title= Imaging proteins inside cells with fluorescent tags |author1=Georgeta Crivat |author2=Justin W. Taraska |journal=Trends in Biotechnology|date=January 2012 |volume= 30|issue=1|pages=8–16|pmc=3246539|pmid=21924508|doi=10.1016/j.tibtech.2011.08.002}}</ref>

===Other Elements===
The expression vector is [[transformation (genetics)|transformed]] or [[Transfection|transfected]] into the host cell for protein synthesis. Some expression vectors may have elements for transformation or the insertion of DNA into the host chromosome, for example the [[agrobacterium|''vir'' genes]] for [[Plant transformation vector|plant transformation]], and [[integrase]] sites for chromosomal integration .

Some vectors may include targeting sequence that may target the expressed protein to a specific location such as the [[periplasmic space]] of bacteria.