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PET, PMAL and GST Vectors

The document discusses various expression vectors, including pET, pMal, and GST vectors, used for producing and purifying proteins in host cells. It details the mechanisms of gene expression control, including the use of T7 RNA polymerase, lac operon elements, and affinity tags for purification. The pET vector system is highlighted for its low copy number and tightly regulated expression, while pMal and GST vectors offer methods for creating fusion proteins for easier purification.

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Gogul Ramnath
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303 views10 pages

PET, PMAL and GST Vectors

The document discusses various expression vectors, including pET, pMal, and GST vectors, used for producing and purifying proteins in host cells. It details the mechanisms of gene expression control, including the use of T7 RNA polymerase, lac operon elements, and affinity tags for purification. The pET vector system is highlighted for its low copy number and tightly regulated expression, while pMal and GST vectors offer methods for creating fusion proteins for easier purification.

Uploaded by

Gogul Ramnath
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PDF, TXT or read online on Scribd
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pET vector

• Expression systems are designed to produce many copies of a desired


protein within a host cell. In order to accomplish this, an expression vector is
inserted into a host cell. This vector contains all of the genetic coding
necessary to produce the protein, including a promoter appropriate to the
host cell, a sequence which terminates transcription, and a sequence which
codes for ribosome binding

• The gene of interest is initially cloned into the pET vector in a bacteria host
that lacks the T7 RNA polymerase gene

• The expression of the gene of interest can be initiated in two possible ways.
The host cells can be infected with phage carrying the T7 RNA polymerase
gene (e.g. λCE6 phage), or more commonly, the pET plasmid can be
transferred into a bacteria host strain whose genome has been engineered
to carry the T7 RNA polymerase gene under the control of the LacUV5
promoter.
• The pET vector exists as a low copy number plasmid in host E. coli, which
reduces leaky expression before induction. The vector utilizes the T7lac
promoter system for strong and tightly controlled gene expression.

• In this system, there is a T7 promoter that can be acted upon by T7 RNA


polymerase to drive high-level expression of the gene of interest.
Additionally, there is a lac operator (LacO) sequence just downstream of the
T7 promoter that can be acted upon by the lac repressor (LacI) protein to
block transcription of the T7 promoter

• The plasmid also carries the natural promoter and coding sequence for LacI.
The LacI protein acts at the LacUV5 promoter in the host cells to repress
expression of the T7 RNA polymerase gene and also functions at the T7lac
promoter on the pET vector to block transcription of the gene of interest by
any T7 RNA polymerase that may be made due to leaky expression.

• Addition of IPTG blocks the inhibitory action of LacI, thereby inducing


expression of T7 RNA polymerase and also removing LacI inhibition of the
gene of interest.
• Although the pET expression system is designed for high-level recombinant protein
expression, the expression level can be reduced by decreasing the amount of IPTG
supplied to host cells. This can be advantageous when expressing proteins with
limited solubility.

• Additionally, the system is able to maintain the gene of interest in a transcriptionally


silent state when T7 RNA polymerase is not present.

• Usually, the host cell for this expression system is a bacteria which has been
genetically engineered to incorporate the gene for T7 RNA polymerase, the lac
promoter and the lac operator in it's genome. When lactose or a molecule similar to
lactose is present inside the cell, transcription of the T7 RNA polymerase is
activated.. Typically, the host cell used is E. coli strain BL(DE3)
• T7 promoter: Drives high-level transcription of the gene of interest when T7
RNA polymerase is present. When placed immediately upstream of a LacO
element, the entire cassette is known as the T7lac promoter.

• LacO: Binding site for LacI. This element inhibits activity of the T7 promoter
when LacI protein is present, preventing leaky expression of the gene of
interest.

• RBS: The ribosome-binding site and translation initiation element from T7


bacteriophage. This allows for efficient production of the protein of interest.

• ORF: The open reading frame of your gene of interest is placed here.

• T7 terminator: Signal sequence to terminate the transcript made from the


gene of interest, preventing run-on transcription.

• Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained


by ampicillin selection in E. coli.

• pBR322 ori: pBR322 origin of replication. Plasmids carrying this origin as


well as the Rop gene exist in low copy numbers in E. coli.
• Rop: Repressor of primer. It encodes a small protein that regulates plasmid
copy number. The presence of the Rop protein, in combination of pBR322
origin of replication on the plasmid, results in low copy numbers of the
plasmid.

• LacI: The E. coli natural promoter and coding sequence for the lac repressor.
pMal vector

• The pMAL-2 vectors provide a method for expressing and purifying a


protein produced from a cloned gene

• The cloned gene is inserted downstream from the malE gene of E. coli,
which encodes maltose binding protein (MBP), resulting in the expression of
an MBP fusion protein

• The vectors express the malE gene fused to the lacZα gene.

• Restriction sites between malE and lacZα are available for inserting the
coding sequence of interest.

• Insertion inactivates the βgalactosidase α-fragment activity of the malE


lacZα fusion, which results in a blue to white color change on Xgal plates
when the construction is transformed into an α-complementing host
• The vectors carry the lacIq gene, which codes for the Lac repressor. This
keeps expression from Ptac low in the absence of IPTG induction

• The pMAL-2 vectors also contain the sequence coding for the recognition site
of a specific protease located just upstream of the polylinker insertion sites.
This allows MBP to be cleaved from the protein of interest after purification
GST Vector - pFN2A (GST) Flexi Vector

• The vector contains a T7 promoter for bacterial or in vitro protein expression of a


protein-coding region.

• The vector appends an N-terminal glutathione-S-transferase (GST) coding


region that can be used to purify the expressed protein.

• The GST tag contains a TEV protease site for removal of the tag after
purification.

• The vector also contains the lethal barnase gene for positive selection of the
insert, an ampicillin-resistance gene for selection of the plasmid and unique SgfI
and PmeI sites that allow easy insertion or transfer of the sequence of interest

• Protein purification with affinity tags such as glutathione S-transferase (GST),


histidine (HIS), and other affinity tags, enables purification of proteins

• GST-tagged fusion proteins can be purified or detected based on the ability of


GST (an enzyme) to bind its substrate, glutathione (GSH).

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