Expression vector
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11 سال پیش
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This lecture explains about the
This lecture explains about the expression vector properties and the uses of expression vector in gene cloning.
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An expression vector, otherwise known as an expression construct, is usually a plasmid or virus designed for protein expression in cells. The 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 encoded by the gene. The plasmid is engineered to contain regulatory sequences that act as enhancer and promoter regions and lead to efficient transcription of the gene carried on the expression vector.[1] The goal of a well-designed expression vector is the production of significant amount of stable messenger RNA, and therefore proteins. Expression vectors are basic tools for biotechnology and the production of proteins, for example insulin which is important for medical treatments of diabetes.
An expression vector has features that any vector may have, such as an origin of replication, a selectable marker, and a suitable site for the insertion of a gene such as the multiple cloning site. The cloned gene may be transferred from a specialized cloning vectors to an expression vector, although it is possible to clone directly into an expression vector. The cloning process is normally performed in Escherichia coli, and vectors used for protein expression 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.
Elements for expression
Further information: Transcription (genetics) and Translation (biology)
An expression vector must have elements necessary for protein expression. These may include a strong promoter, the correct translation initiation sequence such as a ribosomal binding site and start codon, a strong termination codon, and a transcription termination sequence. There are differences in the machinery for protein synthesis between prokaryotes and eukaryotes, therefore the expression vectors must have the elements for expression that is 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 initiates the transcription and is therefore the point of control for the expression of the cloned gene. The promoters used in expression vector are normally inducible, meaning that protein synthesis is only initiated when required by the introduction of an inducer such as IPTG. Protein 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 article: Protein tag
After the expression of the gene product, it is usually 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 purification tag may be added to the cloned gene. This tag could be histidine (His) tag, other marker peptides, or a fusion partners such as glutathione S-transferase or maltose-binding protein. 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. Source of the article published in description is Wikipedia. I am sharing their material. Copyright by original content developers of Wikipedia.
Link- http://en.wikipedia.org/wiki/Main_Page
http://shomusbiology.weebly.com/
Download the study materials here-
http://shomusbiology.weebly.com/bio-m...
An expression vector, otherwise known as an expression construct, is usually a plasmid or virus designed for protein expression in cells. The 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 encoded by the gene. The plasmid is engineered to contain regulatory sequences that act as enhancer and promoter regions and lead to efficient transcription of the gene carried on the expression vector.[1] The goal of a well-designed expression vector is the production of significant amount of stable messenger RNA, and therefore proteins. Expression vectors are basic tools for biotechnology and the production of proteins, for example insulin which is important for medical treatments of diabetes.
An expression vector has features that any vector may have, such as an origin of replication, a selectable marker, and a suitable site for the insertion of a gene such as the multiple cloning site. The cloned gene may be transferred from a specialized cloning vectors to an expression vector, although it is possible to clone directly into an expression vector. The cloning process is normally performed in Escherichia coli, and vectors used for protein expression 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.
Elements for expression
Further information: Transcription (genetics) and Translation (biology)
An expression vector must have elements necessary for protein expression. These may include a strong promoter, the correct translation initiation sequence such as a ribosomal binding site and start codon, a strong termination codon, and a transcription termination sequence. There are differences in the machinery for protein synthesis between prokaryotes and eukaryotes, therefore the expression vectors must have the elements for expression that is 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 initiates the transcription and is therefore the point of control for the expression of the cloned gene. The promoters used in expression vector are normally inducible, meaning that protein synthesis is only initiated when required by the introduction of an inducer such as IPTG. Protein 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 article: Protein tag
After the expression of the gene product, it is usually 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 purification tag may be added to the cloned gene. This tag could be histidine (His) tag, other marker peptides, or a fusion partners such as glutathione S-transferase or maltose-binding protein. 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. Source of the article published in description is Wikipedia. I am sharing their material. Copyright by original content developers of Wikipedia.
Link- http://en.wikipedia.org/wiki/Main_Page
11 سال پیش
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