Question

briefly describe how the GST tag  could be utilized to purify the fusion protein from a cell lysate?

please explain how the gst tag is used not general purification, thank you.

Answer 1

Protein purification with affinity tag such as glutathione S-transferase (GST) , histidine (HIS) and other affinity tags that enables purification of proteins with both known and unknown bio-chemical properties . This methodology has become a widely used research tool for determining the biological function of uncharacterized proteins.

GST is a 211 amino acid protein whose DNA sequence is frequently integrated into expression vector for production of recombinant proteins . The result of expression from this vector is a GST - tagged fusion protein in which the functional GST protein is fused to the N-terminus of the recombinant proteins.

GST-FUSION PROTEIN PURIFICATION-

Glutathione is a tripeptide (Glu-Cys-Gly) that is the specific substrate for glutathione S -transferase (GST) . When reduced glutathione is immobilized through its sulfhydryl group to a solid support , such as cross linked beaded agarose , it can be used to capture pure GST or GST tagged proteins via the enzyme- substrate binding reaction.

The affinity system yields greater than 90% pure GST tagged recombinant protein from crude bacterial or mammalian cell lysate samples.

Glutathione based affinity purification of GST tagged fusion proteins is easily done at either small, medium or large scales to produce microgram,milligram or gram quantities.

At 26 kDa , GST is considerably larger than many other fusion protein affinity tags . For reasons that have not been fully characterized in the literature , the structure of the GST-fusion tag often degrades upon denaturation and reduction for protein gel electrophoresis , as a result electrophoresed samples of GST fusion proteins often appear as a ladder of lower MW bands.