Which vector has glutathione S-transferase tag?

Which vector has glutathione S-transferase tag?

Glutathione S-transferase (GST) is a naturally occurring 26 KDa protein found in eukaryotic cells. The gene from the parasitic helminth Schistosoma japonicum was used in the development of the pGEX vectors (1). This unit describes the use of a GST affinity tag to aid in the purification of recombinant proteins.

What does GST-tag bind to?

glutathione
The GST-tagged protein specifically binds to glutathione immobilized to a matrix (e.g., agarose) and can be easily separated from a cell lysate by a bind-wash-elute procedure.

What is pGEX 2t?

Bacterial vector for expressing GST fusion proteins with a thrombin site.

What is thrombin cleavage?

A thrombin cleavage site (e.g., Leu-Val-Pro-Arg-ll-Gly-Ser; where ll denotes the cleavage site) is widely incorporated within the linker region of fusion or affinity tagged recombinant proteins. The kit contains active thrombin enzyme sufficient to cleave up to 5 mg of the target protein.

What is glutathione S transferase used for?

Glutathione S-transferases (GSTs) are a family of Phase II detoxification enzymes that function to protect cellular macromolecules from attack by reactive electrophiles. Specifically, GSTs catalyse the conjugation of glutathione (GSH) to a wide variety of endogenous and exogenous electrophilic compounds (Figure 1).

Why do we use GST tag?

The GST tag Therefore, this methodology has become a widely used research tool for determining the biological function of uncharacterized proteins. In addition, GST-tagged fusion proteins can be purified or detected based on the ability of GST (an enzyme) to bind its substrate, glutathione (GSH).

How are GST-tagged proteins constructed in pGEX vectors?

GST-tagged proteins are constructed by inserting a gene or gene fragment into the MCS of one of the 13 pGEX vectors. Expression is under the control of the tac promoter, which is induced by the lactose analog isopropyl β-D-thiogalactoside (IPTG). All pGEX vectors are also engineered with an internal lacIq gene.

Why do we use the pgex-6p expression vector?

The pGEX-6P Expression Vectors permit convenient site-specific cleavage and simultaneous purification on Glutathione Sepharose. The pGEX-6P series provides all three translational reading frames linked between the GST coding region and the multiple cloning site.

Why does paer7i not recognize the sequence ctctcgag?

PaeR7I does not recognize the sequence CTCTCGAG. Sticky ends from different PfoI sites may not be compatible. The 1-base overhangs produced by PflFI may be hard to ligate. Sticky ends from different PflFI sites may not be compatible.