GST fusion protein puriication: top question, Top Answer, Biotechnology course

Bacterial Expression Systems Experimental Porotocols in Biotechnology GST-Fusion Proteins GST-Fusion Proteins Protein Expression Systems Protein Purification Purification of Recombinant Proteins

GST is a 211 amino acid protein (26 kDa) whose DNA sequence is frequently integrated into expression vectors for production of recombinant proteins. The result of expression from this vector is a GST-tagged fusion protein in which the functional GST protein (26 kDa) is fused to the N-terminus of the recombinant protein.

How does a GST tag work?

How does GST-tag protein purification work? GST-tag protein purification is an affinity chromatography method. It exploits the high affinity of GST towards reduced Glutathione, its natural substrate.

What is a GST fusion protein?

A gst fusion protein is a protein that is tagged with GST protein. The fusion protein is made by adding the sequence encoding GST to the plasmid expressing your protein of interest. GST has an affinity for GSH making them a good pair for use in chromatography and immunoprecipitation.

What is the benefit of producing a fusion protein that contains a GST tag?

The use of GST as a fusion tag is desirable because it can act as a chaperone to facilitate protein folding, and frequently the fusion protein can be expressed as a soluble protein rather than in inclusion bodies.

What is GST tag and how is it used in the affinity chromatography to purify proteins?

What is GST-tagged protein purification? Recombinant proteins that have Glutathione S-transferase (GST) tags are purified using affinity chromatography. GST binds strongly and specifically to chromatography resins coupled with glutathione.

What is the function of GST in mammalian cells?

GST is a multifunctional enzyme, involved in detoxification processes. This role is achieved by catalytic conjugation of glutathione with a large number of electrophilic toxins and carcinogens. In mammals, GST is found in all tissues and organs.

Why is GST used?

The main objective of incorporating the GST was to eliminate tax on tax, or double taxation, which cascades from the manufacturing level to the consumption level. For example, a manufacturer that makes notebooks obtains the raw materials for, say, Rs. 10, which includes a 10% tax. This means that they pay Rs.

Why does GST bind to glutathione?

Specifically, the function of GSTs in this role is twofold: to bind both the substrate at the enzyme’s hydrophobic H-site and GSH at the adjacent, hydrophilic G-site, which together form the active site of the enzyme; and subsequently to activate the thiol group of GSH, enabling the nucleophilic attack upon the …

Can you Dimerize GST?

Another issue with GST is its propensity to dimerize. Native GST exists as a homodimer, and when fused with a target protein that can also oligomerize, the resulting fusion can form large complexes that are not easily eluted from the bound glutathione resin.

How do I remove GST tag from protein?

Thrombin or Factor Xa can be removed from the protein of interest in one step using a HiTrap™ Benzamidine FF (high sub) column in series after the GSTrap™ column. In this process, the cleaved, tagged protein and thrombin or Factor Xa is washed from the GSTrap™ column onto the HiTrap™ Benzamidine FF (high sub) column.

Why does GST bind to resin?

GST•Bind™ Resin utilizes an 14-atom spacer arm to covalently attach reduced glutathione via a sulfide linkage. The high degree of substitution of glutathione ensures a high binding capacity with yields of glutathione S-transferase (GST) fusion proteins of 5 to 8 mg/ml settled resin.

What is GST affinity chromatography?

Glutathione Affinity is an efficient method for single-step purification of proteins fused to a GST (glutathione S-transferase) tag. GST can be expressed as a soluble protein in the E. coli cytoplasm in high amounts and with full enzymatic activity.

How does GST tag increase solubility?

Because GST folds rapidly into a stable and highly soluble protein upon translation, inclusion of the GST tag often promotes greater expression and solubility of recombinant proteins than expression without the tag.